Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFICATION
PHYSIOLOGICALLY ACTIVE PROTEIN ORIGINATING IN MAMMALS
Technical Field
The present invention relates to a novel physiologically
active protein originating in mammals, a DNA encoding said
protein, and an antibody reactive with said protein.
~~ackground Art
A so-called geriatric disease, which is regarded as a
current disease in high living standard society, includes
arteriosclerosis as well as hypertension and diabetes.
Important measures for preventing these diseases are not only
development of therapeutic methods but also daily life control.
Arteriosclerosis begins with pathological changes (for
example, ( 1 ) invasive growth of smooth muscle cells into inner
membrane,(2)qualitative and quantitative changesof collagen,
elastin, and acidic mucopolysaccharides, and (3) cell foaming
by lipid accumulation in the cytoplasm of grown smooth muscle
cells and macrophages implanting tissues) occurring in inner
membrane of artery. As the result of such pathological changes,
( 1 ) foam cells found in the inner membrane produces fat spots
on the surface of the inner membrane, (2) lipid accumulates
between tissues (deep part of midmembrane) and the inner
membrane surface is covered with thick glass-like membrane,
accompanied by fibrous growth and calcification, and (3)
bleeding and necrosis occur in tissues to cause combined
pathological changes involving thrombogenesis, calcification,
and deposition of lipid crystals. Such pathological changes,
in time, distribute in artery of a whole body and narrow the
_. .cavity of the artery. In addition, the site of pathological
changes becomes bursal and the vascular wall loses elasticity,
thereby hardening blood vessels. The vessels then wind, and
normal blood flow is inhibited.
Epidemiological studies so far have illustrated age
y i
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(about thirties or more), hypercholesterolemia, hypo-HDL-
cholesterolemia, systolic hypertension, obesity, hemoglobin
high value, and diabetes as risk factors of the onset of
arteriosclerosis. Dynamics of in vivo factors inducing the
onset include secretion of adrenalin, increase of thromboxane
A2, decrease of prostacyclin, increase of serum peroxylipid,
increase of free fatty acid, increase of platelet, increase
of fibrinogen, increase of blood coagulation factors (XII and
XIII), decrease of tissue plasmin, increase of prostaglandin,
decrease of antithrombin III, increase of serum LDL, decrease
of serum HDL, increase of insulin, and increase of renin.
Studies so far have revealed only that multiple
conditions, for example, physical conditions such as age and
obesity, complication with other diseases, and abnormalities
of the dynamics of many in vivo factors complicatedly are
related to each other to cause arteriosclerosis.
Treatments of arteriosclerosis are divided with their
purpose into (1) preventive treatments to retract
arteriosclerosis and to prevent the onset of arteriosclerosis
by correcting lifestyle and physical abnormalities such as
obesity (for example, diet therapy and therapeutic exercise)
and (2) chemotherapy or surgical therapy to remove vessel
occlusion symptoms occurring with the progress of
arteriosclerosis or to prevent the onset of vessel cavity
occlusion symptoms by thrombus or embolus,.
Since particular decisive causes of arteriosclerosis are
unclear, only symptomatic treatment by chemotherapy is
currently possible. For example, ,Q blocker is applied when
the enhancement of a catecholamine derivative such as adrenalin
is suspected as the cause, eicosapentaenoic acid is applied
for a prostaglandin derivative, vitamin E is applied for
peroxylipid., and urokinase is applied for thrombus. No
effective pharmaceuticals for treating the arteriosclerosis
have been provided yet.
In the surgical therapy for arterial occulsion,
percutaneous.transluminal coronary angioplasty (PTCA) based
.
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on the observation by angiography prevails clinically as an
effective means to enlarge vessel cavity. PTCA has remarkably
progressed and prevailed since it was clinically applied by
Gruntzig for the first time in 1977, and the number of the
operation has rapidly increased in ,7apan.
PTCA is the method in which the occlusion ( constriction )
site is enlarged by inserting a thin catheter with a balloon
at the tip in a thick catheter into the coronary artery occlusion
site and by expanding the balloon.
However, in cavity enlargement by PTCA, restenosis occurs
at the operation site of the artery in about 30 to 50% of the
cases within a few months after the operation, and this
restenosis is a major drawback of PTCA.
The restenosis has been thought to occur by the
amplification of neonatal inner membrane proliferation based
on the repair reaction of the injury site of the vascular wall,
which has been inevitably caused by the enlargement of the
occlusion site by PTCA. Although chemotherapy has been tried
for preventing this restenosis, almost no effective drugs have
been reported so far.
As mentioned above, at present, a method for the complete
treatment and prevention of arteriosclerosis comprising the
prevention of the recurrence of arteriosclerosis and the
occurrence of restenosis has not established. It is thus
desired to clarify the cause of the onset and progress of
arteriosclerosis and to develop a method for the effective
treatment and prevention thereof, and therapeutic and
preventive drugs.
Coronary artery restenosis occurring after PTCA is
regarded as a clinical model of arteriosclerosis from
pathological viewpoints such as neonatal inner membrane
proliferation or intimal thickening. Therefore, to diagnose
the tissue characteristics of the vascular wall at the
restenosis site after PTCA and to elucidate the difference
between the characteristics and those of normal vascular wall
by comparing them pathologically and at the gene level are
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effective to identify the cause and factors of restenosis, and
further, arteriosclerosis.
In such comparative studies, a useful method for
comparison and examination at the gene level using the genetic
engineering technique is called differential display method
(Nucleic Acids Research, Vo1.21, No. l8, pp.4272-4280 (1993);
and Science, Vo1.257, pp.967-971 (1992)):
Specifically, PCTA is applied to the coronary artery of
a large mammal such as a rabbit, the expression patterns of
genes in the inner membrane tissue at the PTCA site are examined
by differential display method, and they are compared with the
gene expression patterns in the inner membrane tissue without
PCTA, to thereby identify genes specifically or increasingly
expressed after PTCA.
Disclosure of the Invention
Genes that express specifically or increasingly after
PTCA and proteins derived from said genes may be closely related
to arteriosclerosis and restenosis. The present invention
provides pharmaceuticals and methods for preventing and
treating arteriosclerosis and restenosis by identifying genes
and proteins expressing specifically in arteriosclerosis and
coronary artery restenosis.
As the result of studies on the analyses of genes specific
to arteriosclerosis and/or coronary artery restenosis, the
present inventors have discovered genes encoding two novel
proteins (clone BA0306 and BA2303) that express increasingly
at the comparatively early stage (day 1 to 7) after PTCA and
completed the present invention.
The two novel protein-encoding genes of the present
invention, whose characteristics are mentioned below, are
expressed specifically after PTCA, and are thought to be genes
involved in onset and progress of arteriosclerosis and/or
coronary artery restenosis.
Clone BA0306 has the following characteristics.
( 1 ) Its increased expression is observed on day 1 to 7 after
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PTCA of coronary artery (the peak is observed on day 4).
( 2 ) Northern blotting reveals the expression of the mRNA as
about 3.5 k and about 4.4 k bands in various human tissues.
(3) It has ten putative transmembrane regions.
5 (4) It has amino acid sequence homology with S. cerevisiae
oxidative stress resistance protein, S. cerevisiae
zinc/cadmium resistance protein, heavy metal ion resistance
protein, and so on.
(5) The molecules derived from humans and rabbits have the
i0 amino acid sequences of SEQ ID NO: 10 and 8, respectively. The
molecule derived from mice has the amino acid sequence of SEQ
ID NO: 28.
Judging from these characteristics, clone BA0306 is
thought to inhibit active oxygen such as nitrogen monoxide ( NO ) ,
which is involved in the progress of arteriosclerosis and/or
restenosis.
Clone BA2303 has the following characteristics.
(1) Its increased expression is observed from day 1 after
PTCA of coronary artery, and the expression continues until
day 7 with the maximum expression on day 2 to 4.
( 2 ) Northern blotting reveals the expression of the mRNA as
about 3.9 k and about 2.1 k bands in various human tissues.
(3) It has seven putative transmembrane regions.
( 4 ) The molecules derived from humans and mice have the amino
acid sequences of SEQ ID NO: 4 and 6, respectively. The molecule
derived from rabbits has the amino acid sequence of SEQ ID NO:
2.
Judging from these characteristics, clone BA2303 is
thought to be a GTP binding protein ( G protein ) -coupled receptor
that transmits a specific signal through intracellular G
protein to an effector on the plasma membrane or the surface
of the cytoplasm by binding to an in vivo ligand involved in
the onset or progress of arteriosclerosis and/or restenosis.
Therefore, the genes (DNAs ) , proteins, or their fragments
of the present invention and antibodies or a portion of them
reactive with the proteins of the present invention are
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extremely useful for developing drugs for treatment and
prevention of arteriosclerosis and for treatment and
prevention of restenosis after PTCA for artery occlusion
symptom and so on, targeting said genes or protein molecules .
In addition, the DNAs of the present invention themselves are
useful as antisense pharmaceuticals, extracellular region
fragments of said proteins, for example, as soluble receptor
pharmaceuticals, and said antibodies and a portion of them as
antibody pharmaceuticals.
Genes (DNAs), proteins, and antibodies of the present
invention are useful as reagents for searching proteins
(ligands) interacting with the proteins of the present
invention, thereby elucidating the function of said ligands,
and developing therapeutic drugs targeting said ligands.
Based on the genetic information of the rabbit- or
mouse-derived DNA, one embodiment of DNAs of the present
invention, model animals (knockout animals) can be produced
by disrupting (inactivating)the endogenous gene corresponding
to the DNA. Similarly, transgenic animals can be produced as
model animals by introducing the human-derived DNA, one
embodiment of DNAs of the present invention, into nonhuman
mammals such as mice. Function of genes and proteins of the
present invention can be elucidated by analyzing the physical,
biological, pathologic, and genetic characteristics of these
model animals.
Moreover, by mating the model animals whose endogenous
gene is thus disrupted with the transgenic animals, model
animals that have only the human-derived gene of the present
invention can be produced. By administering drugs (compounds,
antibodies, and so on) targeting the introduced human gene to
these model animals, the therapeutic effect of the drug can
be estimated.
Namely, the present invention provides the DNAs,proteins,
expression vectors, transformants, antibodies, pharmaceutical
compositions, transgenic mice, and knockout, mentioned below.
( 1 ) A DNA encoding a protein having the amino acid sequence
r
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of SEQ ID NO: 4.
(2) A DNA encoding a protein fragment comprising the
extracellular region of a protein having the amino acid sequence
of SEQ ID NO: 4.
( 3 ) A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 97 to 1419 of the nucleotide sequence of
SEQ ID NO: 3.
( 4 ) A DNA hybridizing with a DNA having the nucleotide sequence
of SEQ ID NO: 3 under stringent conditions.
(5) A protein having the amino acid sequence of SEQ ID NO:
4 or an amino acid sequence substantially the same as said amino
acid sequence.
(6) A protein fragment comprising the extracellular region
of a protein having the amino acid sequence of SEQ ID NO: 4
or an amino acid sequence substantially the same as said amino
acid sequence.
( 7 ) A fusion protein between the extracellular region of the
protein of (5) and the constant region of the heavy chain of
human immunoglobulin (Ig) or a portion of the constant region.
( 8 ) An expression vector comprising the DNA of any one of ( 1 )
to (4).
(9) A transformant carrying the expression vector of (8).
( 10 ) An antibody or its portion reactive with the protein of
(5) or the protein fragment of (6).
( 11 ) The antibody or its portion of ( 10 ) , wherein the antibody
is a monoclonal antibody.
(12) A pharmaceutical composition comprising the protein
fragment of (6) or the fusion protein of (7) and a
pharmaceutically acceptable carrier.
( 13 ) A pharmaceutical composition comprising the antibody or
its portion of ( 10 ) or ( 11 ) and a pharmaceutically acceptable
carrier.
( 14 ) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 10.
(15) A DNA encoding a protein fragment comprising the
extracellular region of a protein having the amino acid sequence
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of SEQ ID NO: 10.
(16) A DNA having a nucleotide sequence corresponding to
nucleotide residues 1 to 1785 of the nucleotide sequence of
SEQ ID NO: 9.
(17) A DNA hybridizing with a DNA having the nucleotide
sequence of SEQ ID NO: 9 under stringent conditions.
(18) A protein having the amino acid sequence of SEQ ID N0:
or an amino acid sequence substantially the same as said
amino acid sequence.
10 ( 19 ) A protein fragment comprising the extracellular region
of a protein having the amino acid sequence of SEQ ID NO: 10
or an amino acid sequence substantially the same as said amino
acid sequence.
( 20 ) A fusion protein comprising the extracellular region of
the protein of ( 18 ) and the constant region of tl~e heavy chain
of human immunoglobulin (Ig) or a portion of the constant
region.
(21) An expression vector comprising the DNA of any one of
(14) to (17).
( 22 ) A transformant carrying the expression vector of ( 21 ) .
( 23 ) An antibody or its portion reactive with the protein of
(18) or the protein fragment of (19).
( 24 ) The antibody or its portion of ( 23 ) , wherein the antibody
is a monoclonal antibody.
(25) A pharmaceutical composition comprising the protein
fragment of (19) or the fusion protein of (20) and a
pharmaceutically acceptable carrier.
(26) A pharmaceutical composition comprising the antibody or
its portion of ( 23 ) or ( 24 ) and a pharmaceutically acceptable
carrier.
(27) A transgenic mouse in which the human-derived DNA
comprising a DNA having a nucleotide sequence corresponding
to nucleotide residues 97 to 1419 of the nucleotide sequence
of SEQ ID NO: 3 is integrated into an endogenous gene of said
mouse.
(28) A transgenic mouse in which the human-derived DNA
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comprising a DNA having a nucleotide sequence corresponding
to nucleotide residues 1 to 1785 of the nucleotide sequence
of SEQ ID NO: 9 is integrated into an endogenous gene of said
mouse.
(29) A knockout mouse whose endogenous gene encoding a
mouse-derived protein having the amino acid sequence of SEQ
ID NO: 6 is inactivated so that said protein is not produced.
(30) A knockout mouse whose endogenous gene encoding a
mouse-derived protein comprising the amino acid sequence of
SEQ ID NO: 28 is inactivated so that said protein is not
produced.
In the following, the present invention is explained in
detail by clarifying the meanings of terms used in the present
application and the general production methods of proteins,
protein fragments, fusion proteins, DNAs, antibodies,
transgenic mice, and knockout mice of the present invention.
A "protein" of the present invention means a protein and
its fragment derived from mammals such as humans, rabbits, and
mice, and preferably, a human-derived protein and its fragment.
Particularly preferable examples are ( 1 ) a protein having
the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence
substantially the same as said amino acid sequence, ( 2 ) a
protein fragment comprising the extracellular region of a
protein having the amino acid sequence of SEQ ID NO: 4 or an
amino acid sequence substantially the same as said amino acid
sequence, ( 3 ) a protein having the amino acid sequence of SEQ
ID NO: 10 or an amino acid sequence substantially the same as
said amino acid sequence, and (4) a protein fragment comprising
the extracellular region of a protein having the amino acid
sequence of SEQ ID NO: 10 or an amino acid sequence substantially
the same as said amino acid sequence.
The term "extracellular region" used herein is explained
below. A transmembrane protein such as a G protein-coupled
receptors or cell surface molecule connects with the membrane
through the hydrophobic peptide region penetrating the lipid
bilayer of the membrane once or several times and has structure
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composed of three main regions, that is, extracellular region,
transmembrane region, and cytoplasmic region. Such a
transmembrane protein exists as a monomer, homodimer,
heterodimer, or oligomer with another chain ( s ) having the same
5 or different amino acid sequence.
The term "extracellular region" used herein means the
partial structure (partial sequence) existing outside of the
membrane that holds the transmembrane protein as mentioned
above among the whole structure of said membrane protein. In
10 other words, it corresponds to the region excluding the region
incorporated into the membrane (transmembrane region) and the
region existing in the cytoplasm following the transmembrane
region (cytoplasmic region). If desired, one to five amino
acids derived from the amino acids constituting the
transmembrane and/or cytoplasmic region can be added to the
N- terminus and/or C- terminus of the extracellular region in
the present invention.
Here, "having substantially the same amino acid sequence"
means to include a protein having an amino acid sequence where
multiple amino acids, preferably 1 to 10 amino acids,
particularly preferably 1 to 5 amino acids, in the amino acid
sequence shown in SEQ ID NO: 4 or 10, are substituted, deleted,
and/or modified, and a protein having. an amino acid sequence
where multiple amino acids, preferably 1 to 10 amino acids,
26 particularly preferably 1 to 5 amino acids, are added to said
amino acid sequence, as far as the protein has substantially
the same biological properties as the protein having said amino
acid sequence.
Alphabetical triplet or single letter codes used to
represent amino acids in the present specification or figures
mean amino acids asfollows. (Gly/G) glycine, (Ala/A)alanine,
(Val/v) valine, (Leu/L) leucine, (Ile/I) isoleucine, (Ser/s)
serine, (Thr/T) threonine, (Asp/D) aspartic acid, (Glu/E)
glutamic acid, (Asn/N) asparagine, (Gln/Q) glutamine, (Lys/K)
lysine,(Arg/R)arginine, (Cys/C)cysteine,(Met/M)methionine,
(Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W) tryptophane,
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(His/H) histidine, (Pro/P) proline.
"The constant region or a portion of the constant region
of human immunoglobulin (Ig) heavy chain" used herein means
the constant region or the Fc region of human-derived
immunoglobulin heavy chain (H chain) as described, or a portion
of them. The immunoglobulin can be any immunoglobulin
belonging to any class and any subclass. Specifically,
examples of the immunoglobulin are IgG (IgGl, IgG2, IgG3, and
IgG4 ) , IgM, IgA ( IgAl and IgA2 ) , IgD, and IgE. Preferably, the
immunoglobulin is IgG (IgGl, IgG2, IgG3, or IgG4), or IgM.
Examples of particularly preferable immunoglobulin of the
present invention are those belonging to human-derived IgG
(IgGl, IgG2, IgG3, or IgG4).
Immunoglobulin has a Y-shaped structural unit in which
four chains composed of two homologous light chains (L chains )
and two homologous heavy chains ( H chains ) are connected through
disulfide bonds (S-S bonds). The light chain is composed of
the light chain variable region (VL) and the light chain
constant region ( CL ) . The heavy chain is composed of the heavy
chain variable region ( VH ) and the heavy chain constant region
(CH).
The heavy chain constant region is composed of some
domains having the amino acid sequences inherent in each class
(IgG, IgM, IgA, IgD, and IgE) and each subclass (IgGl, IgG2,
IgG3, and IgG4, IgAl, and IgA2).
The heavy chain of IgG (IgGl, IgG2, IgG3, and IgG4) is
composed of VH, CH1 domain, hinge region, CH2 domain, and CH3
domain in this order from N terminus.
Similarly, the heavy chain of IgGl is composed of VH,
C y 11 domain, hinge region, C y 12 domain, and C y 13 domain in this
order from N terminus . The heavy chain of IgG2 is composed of
VH, C y21 domain, hinge region, C y22 domain, and C yZ3 domain
in this order from N terminus. The heavy chain of IgG3 is
composed of VH, C y,l domain, hinge region, C y,2 domain, and
C y,3 domain in this order from N terminus. The heavy chain
of IgG4 is composed of VH, C y,l domain, hinge region, C y,2
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domain, and C y,3 domain in this order from N terminus.
The heavy chain of IgA is composed of VH, C al domain,
hinge region, C a 2 domain, and C a 3 domain in this order from
N terminus.
Similarly, the heavy chain of IgAl is composed of VH,
C a 11 domain, hinge region, C a 12 domain, and C a 13 domain in this
order from N terminus . The heavy chain of IgA2 is composed of
VH, C aZl domain, hinge region, C a22 domain, and C az3 domain
in this order from N terminus.
The heavy chain of IgD is composed of VH, C ~1 domain,
hinge region, C ~ 2 domain, and C ~ 3 domain in this order from
N terminus.
The heavy chain of IgM is composed of VH, C,ul domain,
C,c,C2 domain, C,(,c3 domain, and C,u4 domain in this order from
N terminus and have no hinge region as seen in IgG, IgA, and
IgD.
The heavy chain of IgE is composed of VH, C ~1 domain,
C ~2 domain, C E3 domain, and C E4 domain in this order from
N terminus and have no hinge region as seen in IgG, IgA, and
I gD .
If, for example, IgG is treated with papain, it is cleaved
at the slightly N terminal side beyond the disulfide bonds
existing in the hinge region where the disulfide bonds connect
the two heavy chains to generate two homologous Fab, in which
a heavy chain fragment composed of VH and CH1 is connected with
one light chain through a disulfide bond, and one Fc, in which
two homologous heavy chain fragments composed of the hinge
region, CH2 domain, and CH3 domain are connected through
disulfide bonds (See "Immunology Illustrated", original 2nd
ed., Nankodo, pp.65-75 (1992); and "'Focus of Newest Medical
Science 'Recognition Mechanism of Immune System "', Nankodo,
pp.4-7 (1991); and so on).
Namely, "a portion of a constant region of immunoglobulin
heavy chain" of the present invention means a portion of a
constant region of an immunoglobulin heavy chain having the
structural characteristics as mentioned above, and preferably,
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is the constant region without C1 domain, or the Fc region.
Specifically, examples thereof are the region composed of hinge
region, C2 domain, and C3 domain from each of IgG, IgA, and
IgD, and are the region composed of C2 domain, C3 domain, and
C4 domain from each of IgM and IgE. A particularly preferable
example thereof is the Fc region of human-derived IgGl.
The "fusion protein" of the present invention is that
composed of the above-described extracellular region of the
protein of the present invention and a constant region or a
portion of a constant region of human immunoglobulin ( Ig ) heavy
chain. Preferably, it is a fusion polypeptide composed of an
extracellular region of a protein of the present invention and
a portion of a constant region of human IgG heavy chain, and
particularly preferably, it is a fusion polypeptide composed
of an extracellular region of a protein of the present invention
and the region (Fc) composed of a hinge region, CH2 domain,
and CH3 domain of human IgG heavy chain. Moreover, IgGl is
preferable among IgG. In addition, a protein derived from
human, mouse, or rat ( preferably, human ) is preferable as the
protein of the present invention.
The fusion protein of the present invention has the
advantage that the fusion polypeptide can be purified extremely
easily by using affinity column chromatography using the
property of protein A, which binds specifically to the
2b immunoglobulin fragment because the fusion polypeptide of the
present invention has a portion of a constant region (for
example Fc ) of an immunoglobulin such as IgG as mentioned above
as a fusion partner. Moreover, since various antibodies
against the Fc of various immunoglobulin are available, an
immunoassay for the fusion polypeptides can be easily performed
with antibodies against the Fc.
The protein, protein fragment, and fusion protein of the
present invention can be produced not only by recombinant DNA
technology as mentioned below but also by a method well known
in the art such as a chemical synthetic method and a cell culture
method, or a modified method thereof.
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The DNA of the present invention encodes the above-
mentioned protein of the present invention; and includes any
nucleotide sequence that can encode the protein of the present
invention. The DNA preferably encodes a human-derived protein
of the present invention. Specific examples of the DNA are
described below.
( 1 ) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 4 , a protein fragment composed of the extracellular
region of said protein, or a biological analog obtained by
substituting, deleting, and/or modifying multiple amino acids,
preferably 1 to 10 amino acids, particularly preferably 1 to
5 amino acids in the amino acid sequence of said protein or
fragment, or by inserting multiple amino acids, preferably 1
to 10 amino acids, particularly preferably 1 to 5 amino acids,
in said amino acid sequence.
( 2 ) A DNA encoding a protein having the amino acid sequence
of SEQ ID NO: 10, a protein fragment composed of the
extracellular region of said protein, or a biological analog
obtained by substituting, deleting, and/or modifying multiple
amino acids, preferably 1 to 10 amino acids, particularly
preferably 1 to 5 amino acids, in the amino acid sequence of
said protein or fragment, or by inserting multiple amino acids,
preferably 1 to 10 amino acids, particularly preferably 1 to
5 amino acids, in said amino acid sequence.
( 3 ) A DNA hybridizing with a DNA having the nucleotide sequence
of SEQ ID NO: 3 under stringent conditions.
( 4 ) A DNA hybridizing with a DNA having the nucleotide sequence
of SEQ ID NO: 9 under stringent conditions.
Specific examples thereof are (1) a DNA having a
nucleotide sequence corresponding to nucleotide residues 97
to 1419 of the nucleotide sequence of SEQ ID NO: 3, (2) a DNA
comprising a nucleotide sequence corresponding to nucleotide
residues 1 to 1419 of the nucleotide sequence of SEQ ID NO:
3, (3) a DNA having a nucleotide sequence corresponding to
nucleotide residues 1 to 1785 of the nucleotide sequence of
SEQ ID NO: 9, and (4) a DNA comprising a nucleotide sequence
CA 02282557 1999-08-27
corresponding to nucleotide residues 1 to 1785 of the nucleotide
sequence of SEQ ID NO: 9.
The DNA of the present invention comprises either a
genomic DNA or cDNA. In addition, the DNA includes any DNA
5 composed of any codons encoding the same amino acids.
Examples of "stringent conditions" are as follows. When
a probe with 50 or more nucleotides is used and hybridization
is performed in 0.9% NaCl, the standard of temperature where
50% dissociation occurs (Tm) is calculated using the following
10 formula and the temperature for hybridization can be determined
according to the following formula.
Tm = 82.3°C + 0.41 x (G+C) % - 500/n - 0.61 x (formamide) %
(n means the number of the nucleotide of probe).
Temperature = Tm -25°C.
15 In addition, when a probe with 100 or more nucleotides
(G+C = 40 to 50% ) is used, it should be considered that Tm varies
as (1) and (2) mentioned below.
(1) Tm descends by about 1°C per 1% mismatch.
(2) Tm descends by 0.6 to 0.7°C per 1% formamide.
Accordingly, the temperature conditions for the
combination of completely complementary strands can be set as
follows.
(A) 65 to 75°C (formamide not added)
(B) 35 to 45°C (in the presence of 50% formamide)
The temperature conditions for the combination of
incompletely complementary strands can be set as follows.
(A) 45 to 55°C (formamide not added)
(B) 35 to 42°C (in the presence of 30% formamide)
The temperature conditions when a probe with 23 or less
nucleotides is used can be 37°C or can be calculated using the
following formula.
Temperature = 2°C x (the number of A+T) + 4°C x (the number
of C+G) -5°C.
The DNA of the present invention can be a DNA obtained
by any method. For example, the DNA includes complementary DNA
(cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA
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prepared by chemical synthesis, DNA obtained by PCR
amplification with RNA or DNA as a template, and DNA constructed
by appropriately combining these methods.
The DNA encoding the protein of the present invention
can be obtained by the usual method such as a method to clone
cDNA from mRNA encoding the protein of the present invention,
a method to isolate genomic DNA and then splice them, chemical
synthesis and so on.
( 1 ) cDNA can be cloned from the mRNA encoding the protein of
the present invention by, for example, the method described
below.
First, the mRNA encoding the protein of the present
invention is prepared from the above-described tissues or cells
expressing and producing a cell surface molecule (polypeptide)
of the present invention. mRNA can be prepared isolating total
RNA by a known method such as quanidine-thiocyanate method
(Chirgwin et al., Biochemistry, Vo1.18, p5294, 1979), hot
phenol method, or AGPC method, and subjecting it to affinity
chromatography using oligo-dT cellulose or poly-U Sepharose.
Then, with the mRNA obtained as a template, cDNA is
synthesized, for example, by a well-known method using reverse
transcriptase such as the method of Okayama et al. (Mol. Cell.
Biol. Vol.2, p.161 (1982); ibid. Vol.3, p.280 (1983)) or the
method of Hoffman et al. (Gene Vo1.25, p.263 (1983)), and
converted into double-stranded cDNA. A cDNA library is
prepared by transforming E. coli with plasmid vectors, phage
vectors, or cosmid vectors having this cDNA or by transfecting
E. coli after in vitro packaging.
The plasmid vectors used in this invention are not limited
as long as they are replicated and maintained in hosts. Any
phage vectors that can be replicated in hosts can also be used.
Examples of usually used cloning vectors are pUCl9, ~ gtl0,
~ gtl l , and so on . When the vector is applied to immunological
screening as mentioned below, the vector having a promoter that
can express a gene encoding the polypeptide of the present
invention in a host is preferably used.
CA 02282557 1999-08-27
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cDNA can be inserted into a plasmid by, for example, the
method of Maniatis et al. (Molecular Cloning, A Laboratory
Manual, second edition, Cold Spring Harbor Laboratory, p.1.53,
1989 ) . cDNA can be inserted into a phage vector by, for example,
the method of Hyunh et al . ( DNA cloning, a practical approach,
Vol.l, p.49 ( 1985 ) ) . These methods can be simply performed by
using a commercially available cloning kit (for example, a
product from Takara Shuzo). The recombinant plasmid or phage
vector thus obtained is introduced into appropriate host cells
such as a prokaryote ( for example, E. coli : HB101, DH5 a ,
MC1061/P3, etc.).
Examples of a method for introducing a plasmid into a
host are calcium chloride method, calcium chloride/rubidium
chloride method described in Molecular Cloning, A Laboratory
Manual ( second edition, Cold Spring Harbor Laboratory, p. l . 74
(1989)), and electroporation method. Phage vectors can be
introduced into host cells by, for example, a method in which
the phage DNAs are introduced into grown hosts after in vitro
packaging. In vitro packaging can be easily performed with a
commercially available in vitro packaging kit (for example,
a product from Stratagene or Amersham).
The cDNA encoding the protein of the present invention
can be isolated from the cDNA library so prepared according
to the method mentioned above by combining general cDNA
screening methods.
For example, a clone comprising the desired cDNA can be
screened by a known colony hybridization method ( Crunstein et
al. Proc. Natl. Acad. Sci. USA, Vol. 72, p.3961 ( 1975 ) ) or plaque
hybridization method (Molecular Cloning, A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, p.2 .108 ( 1989 ) )
using 'zP-labeled chemically synthesized oligonucleotides as
probes, which are corresponding to the amino acid sequence of
the polypeptide of the present invention. Alternatively, a
clone having a DNA fragment encoding a specific region within
the polypeptide of the present invention can be screened by
amplifying the region by PCR with synthetic PCR primers.
CA 02282557 1999-08-27
18
When a cDNA library prepared using a cDNA expression
vector ( for example, ~lZAPII phage vector) is used, the desired
clone can be screened by the antigen-antibody reaction using
an antibody against the polypeptide of the present invention.
A screening method using PCR method is preferably used when
many clones are subjected to screening.
The nucleotide sequence of the DNA thus obtained can be
determined by Maxam-Gilbert method (Maxam et al. Proc. Natl.
Acad. Sci. USA, Vol. 74, p. 560 ( 1977 ) ) or the dideoxynucleotide
synthetic chain termination method using phage M13 (Sanger et
al. Proc. Natl. Acad. Sci. USA, Vol. 74, pp.5463-5467 ( 1977 ) ) .
The whole or a portion of the gene encoding the polypeptide
of the present invention can be obtained by excising the clone
obtained as mentioned above with restriction enzymes and so
on.
( 2 ) The DNA encoding the polypeptide of the present invention
can be isolated from the genomic DNA derived from the cells
expressing the polypeptide of the present invention as
mentioned above by the following methods. Such cells are
solubilized preferably by SDS or proteinase R, and the DNAs
are deproteinized by repeating phenol extraction. RNAs are
digested preferably with ribonuclease. The DNAs obtained are
partially digested with appropriate restriction enzymes, and
the DNA fragments obtained are amplified with appropriate phage
or cosmid to generate a library. Then, clones having the
desired sequence are detected, for example, by using
radioactively labeled DNA probes, and the whole or a portion
of the gene encoding the protein of the present invention is
obtained from the clones by excision with restriction enzyme
and so on.
( 3 ) The DNA of the present invention can also be chemically
synthesized by the usual method, based on the nucleotide
sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 27.
The present invention also relates to a recombinant
vector comprising the DNA encoding the protein of the present
invention. The recombinant vector of the present invention is
CA 02282557 1999-08-27
19
not limited as long as it can be replicated and maintained or
can autonomously replicate in various prokaryotic and/or
eukaryotic hosts. The vector of the present invention includes
plasmid vectors and phage vectors.
The recombinant vector can easily be prepared by ligating
the DNA encoding the protein of the present invention with a
vector for recombination available in the art ( plasmid DNA and
bacteriophage DNA) by the usual method. Specific examples of
the vectors for recombination used are E. coli-derived plasmids
such as pBR322, pBR325, pUCl2, pUCl3, and pUCl9, yeast-derived
plasmids such as pSHl9 and pSHl5, and Bacillus subtilis-derived
plasmids such as pUB110, pTP5, and pC194. Examples of phages
are a bacteriophage such as ~l phage, and an animal or insect
virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia
virus, and nuclear polyhedrosis virus.
An expression vector is useful for expressing the DNA
encoding the protein of the present invention and for producing
the polypeptide of the present invention. The expression
vector is not limited as long as it expresses the gene encoding
the polypeptide of the present invention in various prokaryotic
and/or eukaryotic host cells and produces this protein.
Examples thereof are pMAL C2, pEF-BOS (Nucleic Acids Res. Vol.18,
p.5322 (1990)), pMElBS (Experimental Medicine: SUPPLEMENT,
"Handbook of Genetic Engineering" (1992)), and so on.
When bacteria, particularly E. coli are used as host cells,
an expression vector is generally comprised of, at least, a
promoter/operator region, an initiation codon, the DNA
encoding the protein of the present invention, termination
codon, terminator region, and replicon.
When yeast, animal cells, or insect cells are used as
hosts, an expression vector is preferably comprised of, at least,
a promoter, an initiation codon, the DNA encoding the protein
of the present invention, and a termination codon. It may also
comprise the DNA encoding a signal peptide, enhancer sequence,
5'- and 3'-untranslated region of the gene encoding the protein
of the present invention, splicing junctions, polyadenylation
CA 02282557 1999-08-27
.site, selectable marker region, and replicon. The expression
vector may also contain, if required, a gene for gene
amplification (marker) that is usually used.
A promoter/operator region to express the polypeptide
5 of the present invention in bacteria comprises a promoter, an
operator, and a Shine-Dalgarno (SD) sequence (for example,
AAGG). For example, when the host is Escherichia, it
preferably comprises Trp promoter, lac promoter, recA promoter,
~l PL promoter, lpp promoter, tac promoter, or the like.
10 Examples of a promoter to express the polypeptide of the present
invention in yeast are PH05 promoter, PGK promoter, GAP promoter,
ADH promoter, and so on. When the host is Bacillus, examples
thereof are SLO1 promoter, SP02 promoter, penP promoter and
so on. When the host is a eukaryotic cell such as a mammalian
15 cell, examples thereof are SV40-derived promoter, retrovirus
promoter, heat shock promoter, and so on, and preferably SV-40
and retrovirus-derived one. As a matter of course, the
promoter is not limited to the above examples. In addition,
to use an enhancer is effective for expression.
20 A preferable initiation codon is, for example, a
methionine codon (ATG).
The commonly used termination codon ( for example, TAG,
TGA, TAA, and so on) is illustrated as a termination codon.
Usually used natural or synthetic terminators are used
as a terminator region.
A replicon means a DNA capable of replicating the whole
DNA sequence in host cells, and includes a natural plasmid,
an artificially modified plasmid (DNA fragment prepared from
a natural plasmid ) , a synthetic plasmid, and so on. Examples
of a preferable plasmids are pBR322 or its artificial
derivatives (DNA fragment obtained by treating pBR322 with
appropriate restriction enzymes ) for E. coli, yeast 2 ,u plasmid
or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198,
pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC
37149, etc. for mammalian cells.
An enhancer sequence, polyadenylation site, andsplicing
CA 02282557 1999-08-27
21
junction that are usually used in the art, such as those derived
from SV40 can be also used.
A selectable marker usually used can be used according
to the usual method. Examples thereof are resistance genes for
antibiotics, such as tetracycline, neomycin, ampicillin, or
kanamycin, and thymidine kinase gene.
Examples of a gene for gene amplification are
dihydrofolate reductase (DHFR) gene, thymidine kinase gene,
neomycin resistance gene, glutamate synthase gene, adenosine
deaminase gene, ornithine decarboxylase gene, hygromycin-
B-phophotransferase gene, aspartate transcarbamylase gene,
etc.
The expression vector of the present invention can be
prepared by continuously and circularly linking at least the
above-mentioned promoter, initiation codon, DNA (gene)
encoding the polypeptide of the present invention, termination
codon, and terminator region, to an appropriate replicon. If
desired, appropriate DNA fragments (for example, linkers,
restriction sites generated with other restriction enzyme),
can be used by the usual method such as digestion with a
restriction enzyme or ligation using T4 DNA ligase.
Transformants of the present invention can be prepared
by introducing the expression vector mentioned above into host
cells.
Host cells used in the present invention are not limited
as long as they are compatible with an expression vector
mentioned above and can be transformed. Examples thereof are
various cells such as natural cells or artificially established
recombinant cells usually used in technical field of the present
invention (for example, bacteria (Escherichia and Bacillus),
yeast (Saccharomyces, Pichia, etc.), animal cells, or insect
cells.
E. coli or animal cells are preferably used. Specific
examples are E. coli (DH5a, TBl, HB101, etc. ), mouse-derived
cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, etc. ), rat-derived
cells, hamster-derived cells (BHK, CHO, etc.), monkey-derived
CA 02282557 1999-08-27
22
cells (COS1, COS3, COS7, CV1, Velo, etc.), and human-derived
cells(Hela,diploid fibroblast-derived cells,HEK293,myeloma,
Namalwa, etc.).
An expression vector can be introduced (transformed
(transduced)) into host cells by known method.
Transformation can be performed, for example, according
to the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vo1.69,
p.2110 (1972)), protoplast method (Mol. Gen. Genet., Vo1.168,
p. 111 ( 1979 ) ) , or competent method ( J. Mol. Biol. , Vol . 56, p.209
( 1971 ) ) when the hosts are bacteria (E. coli, Bacillus subtilis,
etc. ), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA,
Vo1.75, p.1927 (1978)), or lithium method (J. Bacteriol.,
Vo1.153, p.163 (1983)) when the host is Saccharomyces
cerevisiae, the method of Graham (Virology, Vo1.52, p.456
(1973)) when the hosts are animal cells, and the method of
Summers et al. (Mol. Cell . Biol. , Vol . 3, pp.2156-2165 ( 1983 ) )
when the hosts are insect cells.
The protein of the present invention can be produced by
cultivating transformants (in the following this term includes
transductants) comprising an expression vector prepared as
mentioned above in nutrient media.
The nutrient media preferably comprise carbon source,
inorganic nitrogen source, or organic nitrogen source
necessary for the growth of host cells (transformants).
Examples of the carbon source are glucose, dextran, soluble
starch, and sucrose, and examples of the inorganic or organic
nitrogen source are ammonium salts, nitrates, amino acids, corn
steep liquor, peptone, casein, meet extract, soy bean cake,
and potato extract. If desired, they may comprise other
nutrients ( for example, an inorganic salt ( for example, calcium
chloride,sodium dihydrogenphosphate,and magnesium chloride),
vitamins, antibiotics (for example, tetracycline, neomycin,
ampicillin, kanamycin, etc.).
Cultivation is performed by a method known in the art.
Cultivation conditions such as temperature, pH of the media,
and cultivation time are selected appropriately so that the
CA 02282557 1999-08-27
23
protein of the present invention is overproduced.
Specific media and cultivation conditions used depending
on host cells are illustrated below, but are not limited
thereto.
When the hosts are bacteria, actinomycetes, yeasts,
filamentous fungi, liquid media comprising the nutrient source
mentioned above are appropriate. The media with pH 5 to 8 are
preferably used.
When the host is E. coli, examples of preferable media
are LB media, and M9 media (Miller et al. Exp. Mol. Genet.,
Cold Spring Harbor Laboratory, p.431 (1972)). Using these
media, cultivation can be performed usually at 14 to 43 °C for
about 3 to 24 hours with aeration and stirring, if necessary.
When the host is Bacillus, cultivation can be performed
usually at 30 to 40 °C for about 16 to 96 hours with aeration
and stirring, if necessary.
When the host is yeast, examples of media are Burkholder
minimal media (Bostian, Proc. Natl. Acad. Sci. USA, Vo1.77,
p.4505 (1980)). The pH of the media is preferably 5 to 8.
Cultivation can be performed usually at 20 to 35°C for about
l4to 144 hours with aeration and stirring, if necessary.
When the host is an animal cell, examples of media are
MEMmedia containing about 5 to 20% fetal bovine serum (Science,
Vo1.122, p.501 (1952)), DMEM media (Virology, Vol.8, p.396
(1959)), RPMI1640 media (J. Am. Med. Assoc., Vo1.199, p.519
(1967)), and 199 media (Proc. Soc. Exp. Biol. Med., Vo1.73,
p. l ( 1950 ) ) . The pH of the media is preferably about 6 to 8 .
Cultivation can be performed usually at about 30 to 40°C for
about 15 to 72 hours with aeration and stirring, if necessary.
When the host is an insect cell, an example of media is
Grace's media containing fetal bovine serum (Proc. Natl. Acad.
Sci. USA, Vo1.82, p.8404 ( 1985 ) ) . The pH thereof is preferably
about 5to 8. Cultivation can be performed usually at about 20
to 40°C for 15 to 100 hours with aeration and stirring, if
necessary.
The protein of the present invention can be produced as
CA 02282557 1999-08-27
24
a transmembrane protein by cultivating transformants as
mentioned above, in particular animal cells to overexpress the
protein of the present invention on the surface of the cells.
The protein of the present invention can be produced as a soluble
protein fragment such as an extracellular region protein
fragment by preparing the transformants as mentioned above
using the DNA encoding the extracellular region and by
cultivating the transformants to allow them to secrete the
soluble polypeptide into the culture supernatant.
Namely, a culture filtrate ( supernatant ) is obtained by
the method such as filtration or centrifugation of the obtained
culture, and the protein of the present invention is purified
and isolated from the culture filtrate by the usual method
commonly used in order to purify and isolate a natural or
synthetic protein.
Examples of the isolation and purification method are
a method utilizing solubility, such as salting out and solvent
precipitation method, a method utilizing the difference in
molecular weight, such as dialysis, ultrafiltration, gel
filtration, and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, a method utilizing charges, such as ion
exchange chromatography and hydroxylapatite chromatography,
a method utilizing specific affinity, such as affinity
chromatography, a method utilizing the difference in
2b hydrophobicity, such as reverse phase high performance liquid
chromatography, and a method utilizing the difference in
isoelectric point, such as isoelectric focusing.
When the protein of the present invention exists in the
periplasm or cytoplasm of cultured transformants, first, the
fungus bodies or cells are harvested by the usual method such
as filtration or centrifugation and suspended in appropriate
buffer. After the cell wall and/or cell membrane of the cells
and so on are disrupted by the method such as lysis with
sonication, lysozyme, and freeze-thawing, the membrane
fraction comprising the protein of the present invention is
obtained by the method such as centrifugation or filtration.
CA 02282557 1999-08-27
The membrane fraction is solubilized with a detergent such as
Triton-X100 to obtain the crude extract. Finally, the
polypeptide or the polypeptide fragment is isolated and
purified from the crude extract by the usual method as
5 illustrated above.
The "transgenic mouse" of the present invention is a
transgenic mouse wherein the DNA (cDNA or genomic DNA) prepared
as mentioned above encoding the protein of the present invention
derived from animals except mice (non-self protein) have been
10 integrated into its endogenous locus of the mouse. The
transgenic mouse expresses the non-self protein and secretes
the protein into its body.
The transgenic mouse can be prepared according to the
method as usually used for producing a transgenic animal ( for
15 example, see "Newest Manual of Animal Cell Experiment", LIC
press, Chapter 7, pp.361-408, (1990)).
Specifically, for example, embryonic stem cells (ES
cells) obtained from normal mouse blastocysts are transformed
with an expression vector in which the gene encoding
20 human-derived polypeptide of the present invention (i.e.
"human JTT-1 antigen") has been operably inserted. ES cells
in which the gene encoding the human-derived polypeptide of
the present invention has been integrated into the endogenous
gene are screened by the usual method. Then, the ES cells
25 screened are microinjected into a fertilized egg obtained from
another normal mouse ( blastocyst ) ( Proc . Natl . Acad . Sci . USA,
Vo1.77, No. l2, pp.7380-7384 (1980); U.S. Pat. No. 4,873,191).
The blastocyst is transplanted into the uterus of another normal
mouse as the foster mother . Then, founder mice ( progeny mice )
are born from the foster mother mouse. By mating the founder
mice with normal mice, heterogeneic transgenic mice are
obtained. Hy mating the heterogeneic transgenic mice with each
other, homogeneic transgenic mice are obtained according to
Mendel's laws.
"Knockout mouse" of the present invention is a mouse
wherein the endogenous gene encoding the mouse-derived protein
CA 02282557 1999-08-27
26
of the present invention has been knocked out (inactivated).
It can be prepared, for example, by positive-negative selection
method in which homologous recombination is applied ( U . S . Pat .
No. 5, 464, 764; No. 5, 487, 992; No. 5, 627, 059; PrOC. Natl. Acad.
Sci. USA, Vo1.86, pp.8932-8935 (1989); Nature, Vo1.342,
pp.435-438 (1989); etc.).
The "antibody" of the present invention can be a
polyclonal antibody (antiserum) or a monoclonal antibody, and
preferably a monoclonal antibody.
Specifically, it is an antibody reactive to (against,
which binds to) the above-mentioned protein or its fragment
of the present invention.
The antibody of the present invention can be natural
antibodies obtained by immunizing mammals such as mice, rats,
hamsters, guinea pigs, and rabbits with an immunogen (antigen),
such as the protein of the present invention (natural,
recombinant, or synthetic ones), cells expressing the protein
of the present invention, or transformants overexpressing the
designed protein on the surface thereof prepared using
recombinant DNA technology as described above on the cell
surface. The antibody of the present invention also includes
chimeric antibodies and humanized antibodies (CDR-grafted
antibodies ) that can be produced by recombinant DNA technology,
and human antibodies that can be produced using human
antibody-producing transgenic animals.
The monoclonal antibody includes those having any one
isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.
The polyclonal antibody (antisera) or monoclonal
antibody of the present invention can be produced by the known
methods . Namely, a mammal, preferably, a mouse, rat, hamster,
guinea pig, rabbit, cat, dog, pig, goat, horse, or cattle, or
more preferably, a mouse, rat, hamster, guinea pig, or rabbit
is immunized, for example, with an immunogen (antigen)
mentioned above with Freund's adjuvant, if necessary. The
polyclonal antibody can be obtained from the antiserum obtained
from the animal so immunized. In addition, the monoclonal
CA 02282557 1999-08-27
27
antibodies are produced as follows. Hybridomas are prepared
from the antibody-producing cells obtaind from the animal so
immunized and myeloma cells that are not capable of producing
autoantibodies. The hybridomas are cloned, and clones
producing the monoclonal antibodies showing the specific
affinity to the antigen used for immunizing the mammal are
screened.
Specifically, the monoclonal antibody can be produced
as follows. Immunizations are performed by injecting or
implanting once or several times the protein of the present
invention, cells expressing the protein and so on as mentioned
above as an i.mmunogen, if necessary, with Freund~s adjuvant,
subcutaneously, intramuscularly, intravenously, through the
footpad, or intraperitoneally into a mouse, rat, hamster,
guinea pig, or rabbit, preferably a mouse, rat, or hamster
(including a transgenic animal generated so as to produce
antibodies derived from another animal such as the transgenic
mouse producing human antibody). Usually, immunizations are
performed once to four times every one to fourteen days after
the first immunization. Antibody-producing cells are obtained
from the mammal so immunized in about one to five days after
the last immunization.
Hybridomas that secrete a monoclonal antibody can be
prepared by the method of Kohler and Milstein (Nature, Vo1.256,
pp.495-497 (1975)) and by its modified method. Namely,
hybridomas are prepared by fusing antibody-producing cells
contained in a spleen, lymph node, bone marrow, or tonsil
obtained from the mammal immunized as mentioned above,
preferably a spleen, with myelomas without autoantibody-
producing ability, which are derived from, preferably, a mammal
such as a mouse, rat, guinea pig, hamster, rabbit, or human,
or more preferably, a mouse, rat, or human.
For example, mouse-derived myeloma P3/X63-AG8.653(653),
P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Agl4
(Sp2/0, Sp2), PAI, F0, or BW5147, rat-derived myeloma
210RCY3-Ag.2.3., or human-derived myeloma U-266AR1,
CA 02282557 1999-08-27
28
GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be
used as a myeloma used for the cell fusion.
Hybridoma clones producing monoclonal antibodies can be
screened by cultivating hybridomas, for example, in microtiter
plates and by measuring the reactivity of the culture
supernatant in the well in which hybridoma growth is observed,
to the immunogen used for the immunization mentioned above,
for example, by enzyme immunoassay such as RIA and ELISA.
The monoclonal antibodies can be produced from hybridomas
by cultivating the hybridomas in vitro or in vivo such as in
the ascites fluid of a mouse, rat, guinea pig, hamster, or rabbit,
preferably a mouse or rat, more preferably mouse and isolating
the antibodies from the resulting the culture supernatant or
ascites fluid of a mammal.
Cultivating hybridomas in vitro can be performed
depending on the property of cells to be cultured, on the object
of a test study, and on the various conditions of a cultivating
method, by using known nutrient media or any nutrient media
derived from known basal media for growing, maintaining, and
storing the hybridomas to produce monoclonal antibodies in
culture supernatant.
Examples of basal media are low calcium concentration
media such as Ham~Fl2 medium, MCDB153 medium, or low calcium
concentration MEM medium, and high calcium concentration media
such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640
medium, ASF104 medium, or RD medium. The basal media can
contain, for example, sera, hormones, cytokines, and/or
various inorganic or organic substances depending on the
objective.
Monoclonal antibodies can be isolated and purified from
the culture supernatant or ascites fluid mentioned above by
saturated ammonium sulfate precipitation, euglobulin
precipitation method, caproic acid method, caprylic acid
method, ion exchange chromatography (DEAF or DE52), affinity
chromatography using anti-immunoglobulin column or protein A
column.
r
CA 02282557 1999-08-27
29
The "chimeric antibody" of the present invention is a
monoclonal antibody prepared by genetic engineering, and
specifically means a chimeric antibody such as mouse/human
chimeric monoclonal antibody whose variable regions or the
other regions are derived from mouse immunoglobulin and whose
constant regions are derived from human immunoglobulin.
The constant region derived from human immunoglobulin
has the amino acid sequence inherent in each isotype such as
IgG, IgM, IgA, IgD, and IgE. The constant region of the
recombinant chimeric monoclonal antibody of the present
invention can be that of human immunoglobulin belonging to any
isotype. Preferably, it is the constant region of human IgG.
The chimeric monoclonal antibody of the present invention
can be produced, for example, as follows . Needless to say, the
production method is not limited thereto.
A mouse/human chimeric monoclonal antibody can be
prepared, referring to Experimental Medicine: SUPPLEMENT,
Vo1.1.6, No.lO (1988); and examined published Japanese patent
application (JP-B) No. Hei 3-73280. Namely, it can be prepared
by operably inserting CH gene (C gene encoding the constant
region of H chain) obtained from the DNA encoding human
immunoglobulin downstream of active VH genes (rearranged VDJ
gene encoding the variable region of H chain) obtained from
the DNA encoding a mouse monoclonal antibody isolated from the
hybridoma producing the mouse monoclonal antibody, and CL gene
( C gene encoding the constant region of L chain ) obtained from
the DNA encoding human immunoglobulin downstream of active VL
genes (rearranged VJ gene encoding the variable region of L
chain) obtained from the DNA encoding the mouse monoclonal
antibody isolated from the hybridoma, into the same or different
vectors so as for them to be expressed, following by
transforming host cells with the expression vector, and then
by cultivating the transformants.
Specifically, DNAs are first extracted from mouse
monoclonal antibody-producing hybridomas by the usual method,
digested with appropriate restriction enzymes (for example,
r
CA 02282557 1999-08-27
EcoRI and HindIII), electrophoresed (using, for example, 0.7%
agarose gel), and analyzed by Southern blotting. After an
electrophoresed gel is stained, for example, with ethidium
bromide and photographed, the gel is given with marker positions,
5 washed twice with water, and soaked in 0 . 25 M HC1 for 15 minutes .
Then, the gel is soaked in 0.4 N NaOH solution for 10 minutes
with gently stirring. The DNAs are transferred to a filter for
4 hours by the usual method. The filter is recovered and washed
twice with 2xSSC. After the filter is sufficiently dried, it
10 is baked at 75°C for 3 hours . After baking, the filter is treated
with 0.1 x SSC/0.1% SDS at 65°C for 30 minutes. Then, it is
soaked in 3 x SSC/0.1% SDS. The filter obtained is treated with
prehybridization solution in a plastic bag at 65°C for 3 to 4
hours.
15 Next, 'ZP-labeled probe DNA and hybridization solution
are added to the bag and reacted at 65°C about 12 hours . After
hybridization, the filter is washed under appropriate salt
concentration, reaction temperature, and time (for example,
2 x SSC-0.1% SDS, room temperature, l0 minutes). The filter
20 is put into a plastic bag with a little 2 x SSC, and subjected
to autoradiography after the bag is sealed.
Rearranged VDJ gene and VJ gene encoding H chain and L
chain of a mouse monoclonal antibody are identified by Southern
blotting mentioned above. The region comprising the
25 identified DNA fragment is fractioned by sucrose density
gradient centrifugation and inserted into a phage vector ( for
example, Charon 4A, Charon 28, ~lEMBL3, ~EMBL4, etc. ) . E. coli
( for example, LE392, NM539, etc. ) is transformed with the phage
vector to generate a genomic library . The genomic library is
30 screened by plaque hybridization such as Benton-Davis method
(Science, Vo1.196, pp.180-182 (1977))using appropriate probes
(H chain J gene, L chain ( ~c ) J gene, etc. ) to obtain positive
clones comprising rearranged VDJ gene or VJ gene. By making
the restriction map and determining the nucleotide sequence
of the clones obtained, it is confirmed that genes comprising
the des fired, rearranged VH ( VDJ ) gene or VL ( VJ ) gene are
CA 02282557 1999-08-27
31
obtained.
Separately, human CH gene and human CL gene used for
chimerization are isolated. For example, when a chimeric
antibody with human IgGl is produced, C y 1 gene as a CH gene,
and CEC gene as a CL gene, are isolated. These genes can be
isolated from human genomic library with mouse C yl gene and
mouse C~c gene, corresponding to human C yl gene and human C
~c gene, respectively, as probes, taking advantage of high
homology between the nucleotide sequences of mouse
immunoglobulin gene and that of human immunoglobulin gene.
Specifically, DNA fragments comprising human Cic gene
and an enhancer region are isolated from human il Charon 4A
HaeIII-AluI genomic library (Cell, Vo1.15, pp.1157-1174
(1978)), for example, with a 3 kb HindIII-BamHI fragment of
clone Ig146 (Proc. Natl. Acad. Sci. USA, Vo1.75, pp.4709-4713
(1978) ) and a 6.8 kb EcoRI fragment of clone MEP10 (Proc. Natl.
Acad. Sci. USA, Vo1.78, pp.474-478 (1981)) as probes. In
addition, for example, after human fetal hepatocyte DNA is
digested with HindIII and fractioned by agarose gel
electrophoresis, a 5.9 kb fragment is inserted into X1788 and
then human C y 1 gene is isolated with the probes mentioned above.
Using mouse VH gene, mouse VL gene, human CH gene, and
human CL gene so obtained, and taking promoter region and
enhancer region into consideration, human CH gene is inserted
downstream mouse VH gene and human CL gene is inserted
downstream mouse VL gene into an expression vector such as
pSV2gpt or pSV2neo with appropriate restriction enzymes and
DNA ligase by the usual method. In this case, chimeric genes
of mouse VH gene/human CH gene and mouse VL gene/human CL gene
can be respectively inserted in the same expression vector or
in different expression vectors.
Chimeric gene-inserted expression vectors) thus
prepared are introduced into myelomas that do not produce
antibodies, for example, P3X63 ~Ag8 ~ 653 cells or SP210 cells by
protoplast fusion method, DEAF-dextran method, calcium
phosphate method, or electroporation method. The
CA 02282557 1999-08-27
32
transformants are screened by cultivating in media containing
a drug corresponding to the drug resistance gene inserted into
the expression vector and, then, cells producing desired
chimeric monoclonal antibodies are obtained.
Desired chimeric monoclonal antibodies are obtained from
the culture supernatant of antibody-producing cells thus
screened.
The "humanized antibody (CDR-grafted antibody)" of the
present invention is a monoclonal antibody prepared by genetic
engineering and specifically means a humanized monoclonal
antibody wherein a portion or the whole of the complementarity
determining regions of the hypervariable region are derived
from the complementarity determining regions of the
hypervariable region from a mouse monoclonal antibody, the
framework regions of the variable region are derived from the
framework regions of the variable region from human
immunoglobulin, and the constant region is derived from human
a constant region from immunoglobulin.
The complementarity determining regions of the
hypervariable region exists in the hypervariable region in the
variable region of an antibody and means three regions which
directly and complementary binds to an antigen
(complementarity-determining residues, CDR1, CDR2, and CDR3).
The framework regions of the variable region means four
comparatively conserved regions lying upstream, downstream or
between the three complementarity determining regions
(framework region, FR1, FR2, FR3, and FR4).
In other words, a humanized monoclonal antibody means
that in which the whole region except a portion or the whole
of the complementarity determining regions of the
hypervariable region of a nonhuman mammal-derived monoclonal
antibody have been replaced with their corresponding regions
derived from human immunoglobulin.
The constant region derived from human immunoglobulin
has the amino acid sequence inherent in each isotype such as
IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The
CA 02282557 1999-08-27
33
constant region of a humanized monoclonal antibody in the
present invention can be that from human immunoglobulin
belonging to any isotype. Preferably, it is the constant
region of human IgG. The framework regions of the constant
region derived from human immunoglobulin are not particularly
limited.
The humanized monoclonal antibody of the present
invention can be produced, for example, as follows. Needless
to say, the production method is not limited thereto.
For example, a recombinant humanized monoclonal antibody
derived from mouse monoclonal antibody can be prepared by
genetic engineering, referring to unexamined Japanese patent
publication (JP-WA) No. Hei 4-506458 and unexamined Japanese
patent publication (JP-A) No. Sho 62-296890. Namely, at least
one mouse H chain CDR gene and at least one mouse L chain CDR
gene corresponding to the mouse H chain CDR gene are isolated
from hybridomas producing mouse monoclonal antibody, and human
H chain gene encoding the whole regions except human H chain
CDR corresponding to mouse H chain CDR mentioned above and human
L chain gene encoding the whole region except human L chain
CDR correspond to mouse L chain CDR mentioned above are isolated
from human immunoglobulin genes.
The mouse H chain CDR gene ( s ) and the human H chain gene ( s )
so isolated are operably inserted into an appropriate vector
so that they can be expressed. Similarly, the mouse L chain
CDR gene ( s ) and the human L chain gene ( s ) are operably inserted
into another appropriate vector so that they can be expressed.
Alternatively, the mouse H chain CDR gene(s)/human H chain
genes) and mouse L chain CDR gene(s)/human L chain genes)
can be operably inserted into the same expression vector so
that they can be expressed. Host cells are transformed with
the expression vector thus prepared to obtain transformants
producing humanized monoclonal antibody. By cultivating the
transformants, desired humanized monoclonal antibody is
obtained from culture supernatant.
The "human monoclonal antibody" of the present invention
CA 02282557 1999-08-27
34
is immunoglobulin in which the entire regions comprising the
variable and constant region of H chain, and the variable and
constant region of L chain constituting immunoglobulin are
derived from the gene encoding human immunoglobulin.
b The human antibody can be produced in the same way as
the production method of polyclonal or monoclonal antibodies
mentioned above by immunizing, with an antigen, a transgenic
animal which for example, at least human immunoglobulin gene ( s )
have been integrated into the locus of a non-human mammal such
as a mouse by the usual method. For example, a transgenic mouse
producing human antibodies is prepared by the methods described
in Nature Genetics,Vo1.15,pp.146-156(1997); Nature Genetics,
Vol.7, pp. l3-21 (1994); JP-WA Nos. Hei4-504365, International
patent publication No. W094/25585; Nikkei Science, No.6,
pp.40-50 ( 1995 ) ; Nature, Vol . 368, pp. 856-859 ( 1994 ) ; and JP-WA
No. Hei 6-500233.
The "portion of an antibody" used in the present invention
means a partial region of the antibody, preferably monoclonal
antibody of the present invention as mentioned above, and
specifically, means F ( ab' ) ~, Fab' , Fab, Fv (variable fragment
of antbody ) , sFv, dsFv ( disulf ide stabilized Fv ) , or dAb ( single
domain antibody) (Exp. Opin. Ther. Patents, Vol.6, No.5,
pp.441-456 (1996)).
"F(ab')z" and "Fab "' can be produced by treating
immunoglobulin (monoclonal antibody) with a protease such as
pepsin and papain, and means an antibody fragment generated
by digesting immunoglobulin near the disulfide bonds existing
between the hinge regions in each of the two H chains. For
example, papain cleaves IgG upstream of the disulfide bonds
existing between the hinge regions in each of the two H chains
to generate two homologous antibody fragments in which an L
chain composed of VL (L chain variable region) and CL (L chain
constant region), and an H chain fragment composed of VH (H
chain variable region) and CH yl (yl region in the constant
region of H chain) are connected at their C terminal regions
through a disulfide bond. Each of such two homologous antibody
CA 02282557 1999-08-27
fragments is called Fab'. Pepsin also cleaves IgG downstream
of the disulfide bonds existing between the hinge regions in
each of the two H chains to generate an antibody fragment
slightly larger than the fragment in which the two above-
5 mentioned Fab' are connected at the hinge region. This
antibody fragment is called F(ab')z.
The "pharmaceutical composition" of the present
invention comprises any one of the protein, protein fragment,
fusion protein antibody, or portion of an antibody of the
10 present invention as defined above; and a pharmaceutically
acceptable carrier.
The "pharmaceutically acceptable carrier" includes a
excipieut, a diluent, an expander, a decomposition agent, a
stabilizer, a preservative, a buffer, an emulsifier, an
15 aromatic, a colorant, a sweetener, a viscosity increasing agent,
a flavor, a solubility increasing agent, or other additives.
Using one or more of such carriers, a pharmaceutical composition
can be fomulated into tablets, pills, powders, granules,
injections, solutions, capsules, troches, elixirs,
20 suspensions, emulsions, or syrups. The pharmaceutical
composition can be administered orally or parenterally. Other
forms for parenteral administration include a solution for
external application, suppository for rectal administration,
and pessary, prescribed by the usual method, which comprises
25 one or more active ingredient.
The dosage can vary depending on the age, sex, weight,
and symptom of a patient, effect of treatment, administration
route, period of treatment, or the kind of active ingredient
(polypeptide or antibody mentioned above) contained in the
30 pharmaceutical composition. Usually, the pharmaceutical
composition can be administered to an adult in a dose of 10
,u g to 1000 mg (or 10 ,ug to 500 mg) per one administration.
Depending on various conditions, the dosage less than that
mentioned above may be sufficient in some cases, and the dosage
35 more than that mentioned above may be necessary in other cases .
In particular, the injection can be produced by
CA 02282557 1999-08-27
36
dissolving or suspending the antibody in a non-toxic,
pharmaceutically acceptable carrier such as physiological
saline or commercially available distilled water for injection
with adjusting a concentration to 0.1 ,ug antibody/ml carrier
to 10 mg antibody/ml carrier. The injection thus produced can
be administered to a human patient in need of treatment in a
dose of 1 ,u g to 100 mg/kg body weight, preferably 50 ,u g to
50 mg/kg body weight once or more times a day. Examples of
administration route are medically appropriate administration
routes such as intravenous injection, subcutaneous injection,
intradermal injection, intramuscular injection, or
intraperitoneal injection, preferably intravenous injection.
The injection can also be prepared into a non-aqueous
diluent (for example, propylene glycol, polyethylene glycol,
vegetable oil such as olive oil, and alcohol such as ethanol ) ,
suspension, or emulsion.
The injection can be sterilized by filtration with a
bacteria-non-penetrated filter, by mixing bacteriocide, or by
irradiation. The injection can be produced in the form that
is prepared upon use. Namely, it is freeze-dried to be a sterile
solid composition, and can be dissolved in sterile distilled
water for injection or another solvent before use.
The pharmaceutical composition of the present invention
can be used to treat or prevent arteriosclerosis and restenosis
after the treatment of artery occlusion, such as PTCA.
Brief Descri~,tion of the Drawings
Figure 1 is a photograph showing an electrophoresis image
of rabbit BA2303 cDNA samples obtained by RT-PCR.
The numerals indicate days from the exfoliation of the
artery endothelium using a balloon catheter to the removal of
the artery; thus, the figure shows the time course of the cDNA
expression.
Figure 2 is a photograph showing an electrophoresis image
of rabbit BA0306 cDNA samples obtained by RT-PCR.
The numerals indicate days from the exfoliation of the
CA 02282557 1999-08-27
37
artery endothelium using a balloon catheter to the removal of
the artery; thus, the figure shows the time course of the cDNA
expression.
Figure 3 shows a plot of the hydrophobicity and
hydrophilicity of the amino acid residues composing rabbit
BA2303 protein.
Figure 4 shows a plot of the hydrophobicity and
hydrophilicity of the amino acid residues composing human
BA0306 protein.
Figure 5 shows a plot of the hydrophobicity and
hydrophilicity of the amino acid residues composing human
BA2303 protein.
Figure 6 is a photograph showing the result of Northern
blot analysis of the expression of human BA2303 mRNA in various
human tissues.
Figure 7 is a photograph showing the result of Northern
blot analysis of the expression of human BA0306 mRNA in various
human tissues.
Figure 8 shows a plot of the hydrophobicity and
hydrophilicity of the amino acid residues composing mouse
BA2303 protein.
Figure 9 shows the sequence homology at the amino acid
level between HA2303 proteins from rabbit, human, and mouse.
Figure 10 shows the sequence homology at the amino acid
level between BA0306 proteins from rabbit, human, and mouse.
Figure 11 schematically shows the structures of mouse
genomic DNA containing exons that encode mouse BA2303 protein,
and of the targeting vector for knockout mice generation.
Figure 12 schematically shows the structures of mouse
genomic DNA containing exons that encode mouse BA0306 protein,
and of the targeting vector for knockout mice generation.
Best Mode fo_r ImFlementinq~ the Invention
The present invention is illustrated in detail below with
reference to examples, but is not to be construed as being
limited thereto.
CA 02282557 1999-08-27
38
Example 1
Generation of a rabbit model whose aortal endothelium is
detached by PTCA
According to the method described in "Protocols in
6 Circulation Research"(Jikken-IgakuZoukan(1996)Vo1.14 (12),
87 ) , a balloon catheter was inserted into the thoracic artery
of Japanese white rabbits by surgical operation and was inflated
to perform PTCA. The artery including the operation site was
removed at certain periods from day 1 to six months after PTCA.
E»Tdple 2
Preparation of total RNA from removed aortae
The aorta was removed at 1, 2, 4, 7, 14, 23, 30, 54, 112,
and 137 days after PTCA, and total RNA was prepared from the
aortae by the standard method using the TRIZOL reagent (GIBCO
BRL ) .
Also, the aorta was removed from a normal Japanese white
rabbit, which was not subjected to PTCA, and total RNA was
prepared as described.
Exam In a 3
cDNA synthesis
Total RNAs ( each 2 ~,1, 1 ~,g/ml ) sampled with the passage
of time or mRNA samples (each 2 wl, 0.5 ~,g/ml), which were
obtained in Example 2, were dissolved in diethyl pirocarbonate
(DEPC)-treated distilled water (8 wl). Anchor primer (GT15MA,
1 wl, 25 pmol/~,1) was added to make the total volume 10 ~,1,
and the mixture was then incubated 5 min at 65°C . The samples
were placed on ice immediately after completion of the
incubation.
Then, 5x first strand buffer ( 4 ~1, composition: 0 .25 M
Tris-HC1 (pH 7 . 5 ) , 0. 375 M KC1, 0 . 05 M DTT, 0. 015 M MgCl2 ) , 0.1
M DTT ( 2 ~1 ) , 250 ~I dNTP ( 1 ~ul ) , distilled water ( 1 wl ) , and
reverse transcriptase (Superscript, GIBCO BRL, 1 ~1, 200 U/~,1)
were added to make the total volume 20 wl . cDNA was synthesized
by incubating the reaction mixture for 1 hr at 42°C, and then
DEPC-treated water ( 30 ~,1 ) was added to make the f final volume
5 0 ~,1.
CA 02282557 1999-08-27
39
F;xam~ll_e 4
Analysis of the time course of gene expression
The time course of gene expression after PTCA was analyzed
by the standard method using differential display (Nucleic Acid
Research (1993) Vol. 21(18), 4272-4280; Science (1992) Vol.
257, 967-971), and RT-PCR (reverse transcription-polymerase
chain reaction; "PCR and its Application" (Jikken-Igaku Zoukan
(1990) vol. 8(9); "Gene Amplification PCR Method/Principles
and Novel Applications" Kyoritsu-Syuppan (1992)).
One hundred-fold dilution of the cDNA samples ( each time
point) which were prepared in Example 3 was used as a template
for PCR in differential display. Fifty fold dilution was used
for cDNA samples that were synthesized from mRNA (each time
point).
The template cDNA (each 2 ~,1) was mixed with distilled
water (10.75 ~.1), lOx EX Taq buffer (2 ~1), 25 ~,M dNTP (1.5
wl ) , arbitrary primer ( sequence: GATCAATCGC, 1 ~1, 25 pmol/wl ) ,
anchor primer ( 1 wl, 25 pmol/wl ) , EX Taq DNA polymerase ( 0 . 25
wl), and a35S-dATP (1.5 ~,1, 10 mCi/ml, Amersham) to make the
total volume 20 wl. PCR was carried out with a cycle of 95°C
for 3 min, 40°C for 5 min, 72°C for 5 min; 40 cycles of
95°C for
sec, 40°C for 2 min, 72°Cfor 1 min; and a step of 72°C
for
5 min, and then the samples were kept at 4°C.
Each of the resulting PCR products was mixed with stop
25 buffer (5 wl, composition: formamide (30 ml), xylenecyanol (30
mg ) , bromophenol blue ( 10 mg ) , 0 . 5 M EDTA ( 200 wl, ( pH 8 . 0 ) ) ,
and then, 3.5 wl of each resulting mixture was subjected to
sequence gel electrophoresis on a 6% acrylamide gel
( composition ( in 500 ml total ) : urea ( 240 g ) , lOx TBE ( 50 ml ) ,
30 4 0 % acrylamide ( 7 5 ml , a mixture o f 3 8 % monoac rylamide and 2 %
bisacrylamide)). The result showed that there were two bands
whose expression was changed in the time course.
Both bands were excised from the qel, and two DNA
fragments containing the nucleotide sequences described in SEQ
ID NO: 11 (178 bp) and SEQ ID NO: 12 (167 bp) were isolated
according to the standard method ("Gene Engineering Handbook"
CA 02282557 1999-08-27
Jikken-Igaku, Yodosya (1992)). The fragments were named as
BA2303 (SEQ ID NO: 11 ) , and BA0306 ( SEQ ID NO: 12 ) , respectively.
To confirm the expression of the DNAs containing the two
fragments in the time course, RT-PCR was performed using cDNA
5 samples obtained in Example 3 ( each time point ) as a template.
For amplification of BA2303, synthetic DNA fragments
described in SEQ ID NO: 13 and SEQ ID NO: 14 were used as forward
and reverse primers, respectively.
For amplification of BA0306, synthetic DNA fragments
10 described in SEQ ID NO: 21 and SEQ ID NO: 22 were used as forward
and reverse primers, respectively.
Each template cDNA ( 3 wl ) was mixed with l Ox Vogelstein
buffer (2.5 ~1), 2.5 mM dNTP (1.5 wl), forward primer (1 ~,1,
25 pmol/~,1 ) , reverse primer ( 1 ~,1, 25 pmol/wl ) , ~-actin primer
15 mix (each 25 pmol/~1), and EX Taq DNA polymerase (0.2 ~1),
adjusting the total volume to 25 ~ul. RT-PCR was carried out
with a step of 94°C for 2 min; 35 cycles of 94°C for 3 sec,
55°C
for 30 sec, 72°C for 1 min; and a step of 72°C for 3 min, and
then the samples were kept at 4°C.
20 The obtained PCR products were separated by
electrophoresis. The results were shown in Figures 1 (BA2303)
and 2 (BA0306).
It was confirmed that the expression of BA2303 was
increased from day 1 after the vascular endotheliumwas detached
2b by PTCA, reached the maximal level from about day 2 to day 4,
and continued until about day 7. The expression of BA0306 was
detected over a period from day 1 to day 7 after PTCA, with
peak expression at day 4.
Exams a 5
30 Isolation of long strand cDNA
To isolate long strand cDNAs containing the two cDNA
fragments (BA2303 and BA0306 ) obtained in Example 4, RACE (rapid
amplification ends)-PCR was performed (Proc. Natl. Acad. Sci.
USA (1988) Vol. 85, 8998-9002; "PCR Method for Gene
35 Amplification/Principles and Novel Applications" Kyoritsu-
Syuppan (1992)).
CA 02282557 1999-08-27
41
The PCR was performed twice using the Marathon cDNA
Amplification Kit (CLONTECH) and the cDNA fragments obtained
in Example 4 as a template.
BA2303 was amplified by PCR using synthetic DNA primers
described in SEQ ID NO: 15 and SEQ ID NO: 19 ( 1 ) , and with primers
described in SEQ ID N0: 16 and SEQ ID NO: 20 ( 2 ) , and subsequently
using synthetic DNA primers described in SEQ ID NO: 17 and SEQ
ID NO: 19 ( 3 ) , and with primers described in SEQ ID NO: 18 and
SEQ ID N0: 20 (4).
BA0306 was amplified by PCR using synthetic DNA primers
described in SEQ ID NO: 23 and SEQ ID
NO: 19 (1), and with primers described in SEQ ID NO: 24 and
SEQ ID NO: 20 ( 2 ) , and subsequently using synthetic DNA primers
described in SEQ ID NO: 25 and SEQ ID NO: 19 ( 3 ) , and with primers
described in SEQ ID NO: 26 and SEQ ID NO: 20 (4). The above
PCR produced DNAs described in SEQ ID NO: 1 (BA2303) and in
SEQ ID NO: 7 (BA0306).
Analysis of the deduced amino acid sequence by plotting
the hydrophilicity and hydrophobicity and by PSORT program
suggested that BA2303 is a protein having seven transmembrane
regions (Figure 3).
Exa~ryle 6
Isolation of human counterpart genes
The rabbit cDNAs ( BA2303 and BA0306 ) obtained in Example
5 were used as a probe to screen a human cDNA library (Fetal
Brain, STRATAGENE, code:937-227) by colony hybridization
according to
the standard method ("Gene Engineering HandBook" Jikken-Igaku
Zokan, Yodosya, (1992)). Thus, human homologues containing
the nucleotide sequences described in SEQ ID NO: 3 (BA2303)
and in
SEQ ID NO: 9 (BA0306) were obtained.
Analysis of the deduced amino acid sequence of BA0306
protein by plotting the hydrophilicity and hydrophobicity and
by PSORT program suggested that the protein has 10 transmembrane
regions (Figure 4). It is also suggested that human BA2303
CA 02282557 1999-08-27
42
is a protein having seven transmembrane regions as is the rabbit
one obtained in Example 5 (Figure 5).
Using the respective human DNA as a probe, the expression
of mRNA of the two genes in various human tissues was examined
using the Human Multiple Tissue Northern Blot (CLONTECH, code:
#7760-1, #7759-1).
BA2303 mRNA was expressed in various human tissues as
evident as two bands of about 3 . 9 kb and about 2 .1 kb ( Figure
6).
BA0306 mRNA was also expressed in various human tissues
as detected as two bands of about 3.5 kb and about 4.4 kb (Figure
7).
Homology search between known proteins indicated that
human BA0306 has sequence homology at the amino acid level with
S. cerevisiae oxidative stress resistance protein, S.
cerevisiae zinc/cadmium resistance protein, and heavy metal
ion resistance protein, etc.
Exams a 7
Isolation of mouse BA2303 cDNA
As was described in Example 6, rabbit BA2303 gene was
used as a probe for screening a mouse cDNA library (STRATAGENE,
code: 936-309), and the mouse homologue containing the
nucleotide
sequence described in SEQ ID NO: 5 was isolated. The deduced
amino acid sequence of the coding region was described in SEQ
ID NO: 6.
Analysis of the deduced amino acid sequence by plotting
the hydrophilicity and hydrophobicity and by PSORT program
suggested that mouse BA2303 protein has seven transmembrane
regions as do rabbit and human BA2303 (Figure 8).
BA2303 proteins of the present invention, from rabbit,
human and mouse, have a high sequence homology at the amino
acid level between each other (Figure 9).
ExamFle 8
Isolation of mouse BA0306 cDNA
As was described in Example 6, rabbit BA0306 gene was
CA 02282557 1999-08-27
43
used as a probe for screening a mouse cDNA library (STRATAGENE,
code: 936-309), and the mouse homologue containing the
nucleotide
sequence described in SEQ ID N0: 27 was isolated. The deduced
amino acid sequence of the coding region was described in SEQ
ID NO: 28.
BA0306 proteins of the present invention, from rabbit,
human and mouse, have a high sequence homology at the amino
acid level with each other (Figure 10).
ExamFle 9
Preparation of anti-peptide antibody against human BA2303
An oligopeptide (Gln-Asp-Ala-Gln-Gly-Gln-Arg-Ile-
Gly-His-Phe-Glu-Phe-His-Gly) containing amino acid residues
from 35 to 49 in the sequence described in SEQ ID NO: 4 was
synthesized. Two rabbits were immunized three times with
peptide and Freund's complete adjuvant. The rabbit sera
obtained after each immunization were subjected to ELISA using
horse radish peroxidase-conjugated goat anti-rabbit IgG and
microplates having wells coated with the peptide ( 1 ~,g/well ) ,
and the fluorescence intensity was measured at 492 nm to
determine the antibody titers. Titers were determined as
dilution of sera to obtain a fluorescence intensity at 492 nm
not more than 0.2. The result showed that the titers of
antisera taken from a rabbit A were 50-fold or less before
immunization (3 to 5 ml), 30,600-fold after the first
immunization (16 ml), 40,900-fold after the second
immunization (25 ml), and 41,100-fold after the third
immunization ( 23 ml ) , indicating that the titer was increased
with the number of immunization. The titers of antis era from
the other rabbit B were not more than 50-fold before
immunization (3 to 5 ml), 149,200- fold after the first
immunization (25 ml), 327,500-fold after the second
immunization ( 25 ml ) , and 500, 000-fold or more after the third
immunization ( 25 ml ) , indicating that the titer was increased
and that antibody against the peptide was produced.
Next, the forth immunization was performed on both
CA 02282557 1999-08-27
44
rabbits A and B . The titers after the forth immunization were
46,500-fold in rabbit A, and 500,000-fold or more in rabbit
B as was after the third immunization. Then, the antisera
taken from rabbit A after the forth immunization were purified
by affinity chromatography using a column absorbed with the
peptide that had been used as an antigen. The titer of the sera
from rabbit A after purification was 69,800-fold.
xa Fle 10
Preparation of recombinant fusion protein with human BA2303
protein
Fusion proteins of the present invention were prepared
as a fusion protein with maltose binding protein (MBP) using
the expression plasmid pMAL-C2 (New England Bio Labs. (NEB) ),
which contains a DNA encoding MBP. Experimental procedures
were performed according to the manufacturer's instructions
(Catalogue number: #800, 'Protein Fusion & Purification
System' Ver. 3.03, 12/1994 revised) and by the standard method
of recombinant DNA technology.
Using a template of the DNA encoding human BA2303 (SEQ
ID NO: 3), which was cloned in the previous Example, a DNA
containing the nucleotide sequence corresponding to the N
terminal amino acids ( residues 22 ( Gly ) to 171 ( His ) ) , having
EcoRI and HindIII restriction sites at 5' and 3' termini,
respectively, was amplified by PCR according to the standard
method. Oligonucleotides described in SEQ ID NO: 29 and SEQ
ID NO: 30 were used as 5' and 3' primers, respectively. The
above pMAL-C2 expression plasmid (NEB, inserted with a DNA
encoding MBP) was digested with EcoRi and HindIII, and the
resulting fragments were recovered. Using a commercially
available DNA ligation kit, the above PCR products of human
BA2303 were ligated into the pMAL-C2, and the resulting plasmid
was used to transfected E. coli TBl cells. The bacterial
expression plasmid was prepared in a large quantitiy from the
transformed colony. A culture of the transformed colony
(1/100 volume) was inoculated into 1 liter of LB broth
containing ampicillin and glucose, and incubated with shaking
CA 02282557 1999-08-27
until the OD value became up to 0.5. Then, isopropanol-~-
D-thiogalactopyranoside ( IPTG) was added to the culture to the
final concentration of 0.3 mM, and shaking culture was performed
further ( 3 hr ) . The culture was then centrifuged to remove the
5 supernatant, and the precipitated bacteria was resuspended in
cold column buffer (50 ml, composition: 20 mM Tris-HC1, 200
mM NaCl, 1 mM EDTA, and 10 mM mercaptoethanol ) , which was
supplemented with 0.1 M PMSF (50 ~,1, phenylmethylsulfonyl
fluoride) to suppress protease digestion.
10 The following procedures were carried out on ice unless
otherwise noted. The obtained bacteria suspension was
sonicated on ice to disrupt cells. Then, the suspension was
centrifuged (9000 rpm, 15 to 30 min) to recover soluble fraction.
The soluble fraction was diluted with ice-cold column buffer
15 to load on a column.
Amylose resin ( 15 ml, BIORAD ) was packed in a disposable
column (2.5 dia. x 10 cm), washed, and equilibrated with 8
volumes of ice-cold column buffer. The sample was loaded onto
the column using a pump to keep the flow rate 1 ml/min, and
20 washed with ice-cold column buffer.
The fusion protein was eluted and fractionated with
ice-cold column buffer containinglOmM maltose. Each fraction
was separated by SDS-PAGE, and analyzed by western blotting
using antisera against MBP (NEB). Fractions producing a band
25 detected by western blotting at the position approximately
corresponding to that of the full-length fusion protein were
determined to be positive. Next, the positive fractions were
further purified. MBP/BA2303 fusion protein can be digested
by adding 1 mg/ml factor Xa ( 5 ~,1 ) to the solution containing
30 the fusion protein and incubating it for 24 hr. Digestion of
the fusion protein can be determined by SDS-PAGE followed by
western blotting using antisera against MBP.
Example 11
Preparation of antibody against human BA2303 protein
35 Recombinant protein prepared in Example 10, containing
approximately 150 N-terminal amino acids of human BA2303
CA 02282557 1999-08-27
46
protein (residues 22 (Gly) to 171 (His)), was used as an
immunogen. Two rabbits were immunized with the recombinant
protein and Freund's complete adjuvant. The rabbit sera was
subjected to ELISA using horse radish peroxidase-conjugated
goat anti-rabbit IgG and microplates having wells coated with
the peptide (1 ~,g/well), and the fluorescence intensity was
measured at 492 nm to determine the antibody titers . The titer
was determined as dilution of serum to obtain a fluorescence
intensity at 492 nm not more than 0.2. The result showed that
the titer of sera taken from a rabbit was 50-fold or less before
immunization ( 3to 5 ml ) , and 316, 900- fold after immunization
(18 ml), indicating that the titer was increased. The titer
of the sera from the other rabbit was less than 50-fold before
immunization ( 3 to 5 ml ) , and increased to 312, 300-fold after
immunization (23 ml), indicating that antibody against the
recombinant protein was produced.
Example 12
Construction of an expression vector for recombinant human
BA2303
Using a template of the DNA encoding human BA2303 (SEQ
ID NO: 3), which was cloned in the previous Example, a DNA
containing the nucleotide residues 77 to 1419 (containing the
entire open reading frame ( ORF ) ) , having XbaI restriction sites
at both 5' and 3' termini, was amplified by PCR according to
the standard method. Oligonucleotides described in SEQ ID NO:
31 and in SEQ ID NO: 32 were used as 5' and 3' primers,
respectively.
The resulting PCR products were ligated into the XbaI site of
the pcDNA expression plasmid (Invitrogen) using a commercially
available DNA ligation kit to construct an expression vector
far recombinant human BA2303. Higher eukaryotic host cells
such as COS cells can be transfected with the vector, and the
resulting colonies are selected to obtain transfected cells.
Human BA2303 proteins can be expressed abundantly on the cell
surface of the transfected cells by incubating the them in
appropriate medium such as DMEM containing 10% FCS.
CA 02282557 1999-08-27
47
ExamFle 13
Preparation of recombinant rabbit BA0306 protein
Using a template of the DNA encoding rabbit BA0306 (SEQ
ID NO: 7), which was cloned in the previous Example, a DNA
containing the nucleotide residues 2017 (Ile) to 2196 (Met),
having BamHI and SalI restriction sites at 5' and 3' termini,
respectively, was amplified by PCR according to the standard
method. In the amino acid sequence (60 residues) encoded by
the rabbit nucleotide sequence (nucleotides 2017 (Ile) to 2196
(Met ) ) , 58 residues are the same as those in the corresponding
human BA0306 sequence (residues 535 to 594 in SEQ ID NO: 10).
Oligonucleotides described in SEQ ID NO: 33 and in SEQ ID NO:
34 were used as 5' and 3' primers, respectively.
The expression plasmid pQE-32 (QIA expression type IV
construct, QIAGEN) was digested with BamHI and SalI, and then
blunted.
According to the instruction manual for handling pQE-32,
the obtained PCR products were ligated into the blunted ends
of pQE-32 digested with BamHI-SalI using a commercially
available DNA ligation kit. The resulting expression vector
for recombinant human BA0306 was named as pQE-3287-15.
Next, E. coli cells (XL-1 blue MRF') were transformed
with the pQE-3287-15 according to the standard method, and the
transformed colonies were selected ("Gene Engineering
Handbook" Jikken-Igaku Bessatsu, Yodosha (1992) 46-51). A
culture of the transformed cells was inoculated into LB broth
containing ampicillin and glucose, and incubated at 37°C with
shaking, with measuring the OD. Then, IPTG (isopropanol-~-
D-thiogalactopyranoside) was added to the culture to the final
concentration of 1 mM, and shaking culturing was further
performed at 37°C for 4 hrs. The culture was centrifuged to
remove the supernatant, and the precipitated bacteria was
resuspended in column buffer. The suspension was sonicated on
ice to disrupt cells, then centrifuged, and soluble fraction
was recovered. The soluble fraction was diluted with ice-cold
column buffer to load on a column.
CA 02282557 1999-08-27
48
A column was packed with Ni-NTA resin, washed, and
equilibrated with column buffer. The samples were applied on
the column and washed with column buffer. The eluted fractions
were collected, and thus recombinant rabbit BA0306 protein was
obtained.
.xa Fle 1414
Preparation of antibody against human BA0306
Recombinant rabbit BA0306 protein prepared in Example
13 was used as an immunogen. The protein and Freund' s complete
adjuvant were used to immunize chickens . The chicken sera were
subjected to ELISA using horse radish peroxidase-conjugated
anti-chicken IgG and microplates having wells coated with the
recombinant protein (1 ~,g/well), and the fluorescence
intensity was measured to determine the antibody titers. The
result showed that the titer was increased, indicating that
antibody against the ecombinant protein was produced.
Furthermore, rabbit BA0306 protein fragment, which was
used as an immunogen in this example, and the above recombinant
human BA0306 protein were detected by western blotting using
the chicken antisera, indicating that the antisera had a cross
reactivity with human BA0306 protein.
ExamFle 15
Generation of knockout mice of mouse BA2303 gene
A knockout mouse, whose endogenous gene encoding mouse
BA2303 protein was inactivated, was generated as follows.
(1) Construction of a targeting vector
A targeting vector for generation of a knockout mouse,
in which an endogenous gene encoding mouse BA2303 protein was
inactivated (knocked out) by homologous recombination
(Nikkei-Science (1994) May, 52-62), was constructed as
follows.
The cDNA encoding mouse BA2303 protein (SEQ ID NO: 5),
which was cloned in the previous Example, was labeled with 'ZP
by the standard method to obtain a probe used in hybridization.
The probe was used to screen a cosmid mouse genomic DNA library
(~~Labomanual Human Genome Mapping" Hori M., and Nakamura Y.
CA 02282557 1999-08-27
49
edit., Maruzen Syuppan), and thus, a mouse genomic DNA clone
containing exons (E1, E2, and E3) which encode mouse BA2303
protein was isolated. The structure of the genomic DNA was
schematically shown in Figure 11. The genomic DNA was
subcloned into a plasmid, and digested with SacII to remove
the region of 124 by encompassing E1 and the intron between
E1 and E2, and then ligated with an insert of a neomycin
resistance gene of 1143 by (neo, as a positive selection marker) ,
which had been digested with restriction enzymes and blunted.
The plasmid pBluescript II SK ( - ) was digested with SacII,
and ligated with an insert of a thymidine kinase gene (TK, as
a negative selection marker). Then, the resulting
pBluescript II SK(-) was digested with XbaI, and ligated with
an insert of the above mouse BA2303 genomic DNA having a neo
gene insertion.
(2) Transfection of the targeting vector into ES cells
Mouse embryonic stem cells (ES cells ) (Nature ( 1993 ) 362,
255-258; Nature (1987) 326, 292-295), which were cultured in
DMEM containing 15% fetal bovine serum, were trypsinized to
obtain single isolated cells, washed three times in phosphate
buffer, and prepared as a cell suspension of 1 x 10' cells/ml.
The targeting vector was added to the cells (25 ~,g/1 ml cell
suspension), and electroporation was performed with a single
pulse of 350 V/cm ( 25 ~F ) . Then, the ES cells were seeded into
10 cm dishes (1 x 107 cells/dish), cultured for one day in
maintenance medium, and then the medium was replaced with
selection medium (containing 6418 (250 ~,g/ml) and 2 N,M
gancyclovir). The culture was continued with replacing the
medium every two days. On the tenth day after transfection of
targeting vector, 540 neomycin resistant ES clones were
isolated using a micropipet under microscopic observation.
The clones were cultured separately in 24 well plates layered
with feeder cells, and replica of 540 neomycin resistant ES
cells were obtained.
(3) Screening of knockout ES cells
Each neomycin resistant ES clone was examined by PCR
CA 02282557 1999-08-27
whether its endogenous gene encoding mouse BA2303 protein was
inactivated (knocked out) by homologous recombination.
PCR was performed using genomic DNA extracted from each
neomycin resistant ES clone as a template, with two primers
5 designed based on the sequence of the neo gene (SEQ ID NO: 36
and SEQ ID NO: 37) (1) and on the mouse BA2303 genomic DNA
sequence which locates on the flanking region of the BA2303
DNA which was inserted in the targeting vector (SEQ ID NO: 35
and SEQ ID NO: 38) (2). DNA was purified using an automated
10 DNA purification robot (Kubota). The result showed that
desired PCR products were obtained in several clones among the
ES clones examined. Further selection of these clones can be
performed by genomic Southern blotting. Genomic DNA was
extracted from each clone, digested with restriction enzymes,
15 and separated by electrophoresis on an agarose gel. Then, the
DNA was transferred onto a nylon membrane, and subjected to
hybridization using a probe designed based on the genomic
sequence of mouse BA2303. The probe was designed based on the
sequence which locates in the flanking region of the site of
20 homologous recombination, and thus enabled to distinguish
mutated genome from normal one by size. The knockout ES clone
selected in this way was used for generation of knockout mice
as described below.
(4) Generation of knock out mice
25 The above obtained ES cells, having inactivation in the
endogenous gene encoding mouse BA2303 protein as a result of
homologous recombination, were injected into blastocysts
obtained by crossing C57BL6 mice (Japan Charles River) (15
cells/embryo, microinjection). Immediately after
30 microinjection, the blastocysts were implanted into uterines
of ICR mice ( Clea Japan ) , which had undergone pseudopregnancy
treatment two days and half before (10 blastocysts/one side
of the uterine). Thus, desired chimera mice were obtained.
The chimera were crossed with normal C57BL6 mice to obtain
35 agouti mice, whose color is attributed to a gene determining
hair color, originating from ES cells.
CA 02282557 1999-08-27
51
Exa ple 16
Generation of knockout mice of mouse BA0306 gene
(1) Construction of a targeting vector.
A targeting vector for generation of a knockout mouse,
in which the endogenous gene encoding mouse BA0306 protein was
inactivated (knocked out) by homologous recombination
(Nikkei-Science (1994) May, 52-62), was constructed as
follows.
The cDNA encoding mouse BA0306 protein (SEQ ID NO: 27 ) , which
was cloned in the previous Example, was labeled with '2P by the
standard method to obtain a probe used in hybridization. The
probe was used to screen a 129SVJ mouse genomic DNA library
(STRATAGENE), and a mouse genomic DNA clone containing exons
( exon I, II, III, IV, and V ) that encode mouse BA0306 protein
was isolated.
The plasmid pBluescript II SK(-) was digested with Xhol
and HindIII, and ligated with Xhol-HindIII-digested thymidine
kinase gene (TK, as a negative selection marker). Next,
NotI-digested pBluescript II SK(-) was ligated with an insert
of the above mouse BA0306 genomic DNA (exons I to V). Then,
the neomycin resistance gene (neo, as a positive selection
marker ) was digested with BamHI and XhoI, blunted, and ligated
into the Aor5lHI site of the exon V in the mouse BA0306 genomic
DNA. Finally, the resulting pBluescript II SR(-) was digested
with SacII and linealized to use as a targeting vector.
(2) Transfection of the targeting vector into ES cells.
Mouse embryonic stem cells (ES cells, 1 x 108 cells)
(Nature ( 1993 ) 362, 255-258; Nature ( 1987 ) 326, 292-295 ) , which
were cultured in DMEM containing 15% fetal bovine serum, were
trypsinized to obtain single isolated cells, washed three times
in phosphate buffer, and~then prepared as a cell suspension
of 1 x 10' cells/ml. The targeting vector was added to the
( 25 wg/1 ml cell suspension ) , and electroporation was performed
with a single pulse of 350 V/cm (25 ~F) . Then, the ES cells
were seeded into 10 cm dishes (1 x 10' cells/dish), cultured
1 day in maintenance medium, and then the medium was replaced
CA 02282557 1999-08-27
52
with selection medium (containing 6418 (250 ~,g/ml) and 2 ~,iM
gancyclovir). The culture was continued with replacing the
medium every two days. On the tenth day after transfection,
573 neomycin resistant ES clones were isolated using a
micropipet under microscopic observation. The clones were
cultured separately in 24 well plates layered with feeder cells,
and replica of 573 neomycin resistant ES cells were obtained.
(3) Screening of knockout ES cells
Each neomycin resistant ES clone was examined by genomic
Southern blotting whether its endogenous gene encoding mouse
BA0306 protein was inactivated (knocked out) through
homologous recombination.
Genomic DNA was extracted from each neomycin resistant
ES clone, and genomic Southern blotting was performed on EcoRI
digested genomic DNA fragments according to the standard method
using the following probes.
(probe 1)
5' flanking DNA which was amplified using two primers
described in SEQ ID NO: 39 and SEQ ID NO: 40.
(probe 2)
3' flanking DNA which was amplified using two primers
described in SEQ ID NO: 41 and SEQ ID NO: 42.
DNA was purified using an automated DNA purification robot
(Kubota).
If the endogenous gene encoding BA0306 is normally
targeted by the targeting vector, the 5 ~ and 3 ~ flanking genes
encompassing the integrated neo gene can be detected as 7 kb
and 5 kb bands, respectively.
The result showed that desired knockout of the gene was
occurred in three ES clones (named as 0-16-9, 0-22-11, and
0-22-18), which were used for generation of knockout mice as
described below.
(4) Generation of knock out mice
The ES clones obtained above, having inactivation in the
endogenous gene encoding mouse BA0306 protein as a result of
homologous recombination, were microinjected into blastocysts
r
CA 02282557 1999-08-27
53
obtained by crossing C57BL6 mice (Japan Charles River) (15
cells/embryo). Immediately after microinjection, the
blastocysts were transferred to uterines of ICR mice (flea
Japan) (10 blastocysts/one side of the uterine), which had
undergone pseudopregnancy treatment two days and half before.
As a result, desired knockout chimera mice were obtained from
each ES clone as followings.
(clone 0-16-9)
Total number of injected cells: 83
Littermates: 13
Chimera mice: 7
Chimera where contribution to hair color is 80% or more:
2
(clone 0-22-11)
Total number of injected cells: 202
Littermates: 12
Chimera mice: 3
Chimera where contribution to hair color is 80% or more:
3
(clone 0-22-18)
Total number of injected cells: 148
Littermates: 9
Chimera mice: 5
The chimera were crossed with normal C57BL6 mice to obtain
agouti mice whose color is attributed to a gene determining
hair color, originating from ES cells.
Industrial AFylicabilitv
The present invention provides two novel physiologically
active protein molecules (BA0306, and BA2303) having
characteristics described below, which are specifically
expressed in arteriosclerosis or coronary restenosis, and are
predicted to relate closely to the onset and progress of these
diseases; their fragments; a gene (DNA) encoding the protein
molecules; an antibody reactive with the molecule, and its
fragment; and pharmaceutical compositions comprising the above
CA 02282557 1999-08-27
54
protein molecule or the antibody.
[BA0306]
A molecule having the following characteristics, and
presumed to have inhibitory effects on active oxygen species
such as nitrogen monoxide (NO), which has been identified to
be involved in the progress of arteriosclerosis and restenosis .
( 1 ) Its expression is increased from day 1 to day 7 after PTCA
of the coronary aorta (peak at day 4).
( 2 ) Its mRNA is expressed in various human tissues as detected
by Northern blotting as approximately 3 . 5 kb and 4 . 4 kb bands .
(3) Its 10 predicted transmembrane regions.
( 4 ) Its sequence homology at the amino acid level with S .
cerevisiae oxidative stress resistance protein, S. cerevisiae
zinc/cadmium resistance protein, and heavy metal ion
resistance protein, etc.
[BA2303]
A molecule having the following characteristics, and
presumed to be a G protein(GTP binding protein)-coupled
receptor that transduces a specific signal through
intracellular G protein to an effector on the plasma membrane
or in the cytoplasm by binding to an in vivo ligand which is
involved in the onset and progress of arteriosclerosis and
restenosis.
( 1 ) Its expression is increased day 1 after PTCA of the coronary
aorta, reaches the maximum on day 2 to day 4, and continued
until day 7.
( 2 ) Its mRNA is expressed in various human tissues as detected
by Northern blotting as approximately 3 . 9 kb and 2 .1 kb bands .
(3) having seven predicted transmembrane regions.
Therefore, a gene (DNA) or protein of the present
invention or its part, and an antibody reactive with the protein,
or a part of the antibody are extremely useful in developing
the drugs targeting the gene or the protein molecule for
treatment and prevention of arteriosclerosis as well as
restenosis after PTCA of arterial embolism. Also, the DNA
itself is very useful as an antisense medicine, the
CA 02282557 1999-08-27
extracellular domain fragment of the protein is useful as a
soluble receptor medicine, and the antibody or its part is
useful as an antibody medicine.
Furthermore, the gene (DNA), protein, and antibody of -
5 the present invention are useful as a reagent for screening
a protein ( ligand ) interacting with the protein of the invention,
identification of the function of the ligand, and developing
a drug which targets the ligand.
In addition, based on the nucleotide sequence originating
10 from rabbit or mouse, as an embodiment of the DNA of the present
invention, model animals (knockout animals) can be generated
by disrupting (inactivating) a corresponding endogenous gene.
Similarly, trasngenic animals can be generated as a model animal
by introducing human DNA, as an embodiment of the DNA of the
15 present invention, into mammals such as mice except human. It
is possible to identify the functions of the gene and protein
of the invention by analyzing the physical, biological,
pathological, and genetical characteristics of the model
animals.
20 Moreover, it is possible to generate model animals having
a human gene of the invention alone by crossing the model animals,
whose endogenous gene is disrupted, with the transgenic animals .
Thus, it is possible to estimate the therapeutic effects of
a drug which targets the introduced human gene ( compounds , and
25 antibodies, etc.) by administrating the drug into the model
animals .
CA 02282557 1999-08-27
56
Seguence listing
(1) Name of applicant: JAPAN TABACCO INC.
(2) Title of the invention: PHYSIOLOGICALLY ACTIVE PROTEIN ORIGINATING IN
MAMMALS
(3) Reference number: J1-002PCT
(4) Application number:
(5) Filing date:
(6) Country where the priority application was filed and the application
number of the application:
to Japan, No. Hei 9-62259
Japan, Patent Application f f led February 25, 1998 (reference number,
J98-0048)
(7) Priority date: February 28, 1997
(8) Number of sequences: 42
SEQ ID N0: 1:
SEQUENCE LENGTH: 1399
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
2o TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA containing 3~-end nucleotide
sequence)
ORIGINAL SOURCE:
ORGANISM: Oryctolagus
FEATURE:
NAME/KEY: CDS
LOCATION: 1 .. 1158
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 1
3o GTC AGA ATC AAC AAC ATA GCA GTA GCT GTA GGA AAA GAA GCT 42
Val Arg Ile Asn Asn Ile Ala Val Ala Val Gly Lys Glu Ala
1 5 10
AAA CTT TAC CTG TTC CAA GCC CAG GAA TGG CTG AAG CTG CAG 84
Lys Leu Tyr Leu Phe Gln Ala Gln Glu Trp Leu Lys Leu Gln
15 20 25
GAA AGC AGT CAT GAT TAC AGC TGT CAT GAA AAA TTA TCC AAA 126
CA 02282557 1999-08-27
57
Glu SerSerHis AspTyrSer CysHisGluLys LeuSerLys
30 35 40
GCC CAATTGACA ATGACCATG AACCAGAGTGAA CATAATATG 168
Ala GlnLeuThr MetThrMet AsnGlnSerGlu HisAsnMet
45 50 55
ACA GTGTCCCAG ATTCCATCT CCACAAACGTGG CACGTGTTT 210
Thr ValSerGln IleProSer ProGlnThrTrp HisValPhe
60 65 70
TAT GCAGACAAG TATACATGC CGAGTTGACGAG GAGAATTGG 252
ioTyr AlaAspLys TyrThrCys ArgValAspGlu GluAsnTrp
75 80
CAA GTGGAAGAT ATCCCATTT GAAATGGTGTTA CTAAACCCA 294
Gln ValGluAsp IleProPhe GluMetValLeu LeuAsnPro
85 90 95
~5GAT GCTGAAGGA AATCCGTTT GATCATTTTGGT GCTGGAGAA 336
Asp AlaGluGly AsnProPhe AspHisPheGly AlaGlyGlu
100 105 110
TCT GGGTTACAT GAGTTCTTT TTCCTCCTAGTC CTAGTGTAC 378
Ser GlyLeuHis GluPhePhe PheLeuLeuVal LeuValTyr
20 115 120 125
TTT GTGACTGCT TGCATTTAT GCGCAGTCATTG TGGCAGGCT 420
Phe ValThrAla CysIleTyr AlaGlnSerLeu TrpGlnAla
130 135 140
CTT AAGAAAGGA GGGCCCATG CACATGATTCTA AAGGTGCTG 462
25Leu LysLysGly GlyProMet HisMetIleLeu LysValLeu
145 150
ACA ACTGCACTG CTGTTGCAA GCTGGTTCAGCT GTAGCTAAT 504
Thr ThrAlaLeu LeuLeuGln AlaGlySerAla ValAlaAsn
155 160 165
3oTAC ATCCATTTC TCCAGTTAC TCCAAAGATGGA ATCGGGGTA 546
Tyr IleHisPhe SerSerTyr SerLysAspGly IleGlyVal
170 175 180
CCT TTTATGGGA AGCTTGGCA GAATTTTTTGAC ATCGCTTCC 588
Pro PheMetGly SerLeuAla GluPhePheAsp IleAlaSer
35 185 190 195
CAA ATTCAGATG TTATACCTG CTTCTGAGTCTG TGCATGGGC 630
CA 02282557 1999-08-27
58
Gln Ile GlnMetLeu TyrLeuLeu LeuSerLeuCys MetGly
200 205 210
TGG ACC ATAGTCAGG ATGAAGAAG TCTCAAAGCAGA CCTCTC672
Trp Thr IleValArg MetLysLys SerGlnSerArg ProLeu
215 220
CAG TGG GATTCGACC CCTGCCTCC ACTGGCATTGCC GTGTTC714
Gln Trp AspSerThr ProAlaSer ThrGlyIleAla ValPhe
225 230 235
ATT GTC CTGACACAG AGTGTTTTG CTGCTTTGGGAA CAGTTT756
io Ile Val LeuThrGln SerValLeu LeuLeuTrpGlu GlnPhe
240 245 250
GAA GAT ACCGGTCAT CATAGCTCC CATTCACACCAC AACTTA798
Glu Asp ThrGlyHis HisSerSer HisSerHisHis AsnLeu
255 260 265
GCA GGG ATCCTTCTG ATCGTTTTA AGAATTTGCCTG GCATTG840
Ala Gly IleLeuLeu IleValLeu ArgIleCysLeu AlaLeu
270 275 280
TCA TTA GGCTGTGGA CTCTATCAG ATCATCACAGTG GAGAGG882
Ser Leu GlyCysGly LeuTyrGln IleIleThrVal GluArg
285 290
AGC ACA CTCAAAAGG GAGTTCTAC ATCACATTTGCC AAAGGC924
Ser Thr LeuLysArg GluPheTyr IleThrPheAla LysGly
295 300 305
TGT ATC TTATGGTTT TTGTGCCAT CCAAGTCTGGCA TGCATT966
Cys Ile LeuTrpPhe LeuCysHis ProSerLeuAla CysIle
310 315 320
TCT GTC ATTTTTAAT GACTACCAA AGAGATAAGGTT ATTACA1008
Ser Val IlePheAsn AspTyrGln ArgAspLysVal IleThr
325 ~ 330 335
ATA GGT GTTATCCTT GGCCAGTCT GTTGCCATGGTT ATCCTC1050
Ile Gly ValIleLeu GlyGlnSer ValAlaMetVal IleLeu
340 345 350
TAC AGA CTCTTTCTC TCCCACAGT CTATACTGGGAA GTTTCT1092
Tyr Arg LeuPheLeu SerHisSer LeuTyrTrpGlu ValSer
355 360
TCC CTT TCCTCAGTA ACACTACCA CTGACCGTATCG TCTGGA1134
CA 02282557 1999-08-27
59
Ser Leu Ser Ser Val Thr Leu Pro Leu Thr Val Ser Ser Gly
365 370 375
CAC AAA AGC CGC CCT CAT TTC TGA TACTTGATTT CTGTGGAAAA 1178
His Lys Ser Arg Pro His Phe
380 385
GAAAAGTGAA GGGGTTAAAA GAGTGCAATA AGGACCCAAA TACAGTGACT 1228
TTTTTTTCAT ACATTTGGTA TGAAAAATCG AATAGCAAAA GCAGAGCATG 1278
TTTCTGTGAT AACTGCATTT AAGCAGTACC AAAACTGAAC AAAGGTAATA 1328
ACTGAAATGT TTTAAAATAC ATGTAAACAA TAAACTTTCA GGAAATTCTG 1378
~o TTGTTAAAAA AAA~AAAAAAA C 1399
SEQ ID N0:
2:
SEQUENCE 385
LENGTH:
SEQUENCE acid
TYPE:
amino
~5 TOPOLOGY:
linear
MOLECULE
TYPE:
protein
SEQUENCE 2
DESCRIPTION:
SEQ
ID
N0:
Val Arg Ile AsnIleAla ValAlaVal GlyLysGlu Ala
Asn
1 5 10
2o Lys Leu Tyr PheGlnAla GlnGluTrp LeuLysLeu Gln
Leu
20 25
Glu Ser Ser AspTyrSer CysHisGlu LysLeuSer Lys
His
30 35 40
Ala Gln Leu MetThrMet AsnGlnSer GluHisAsn Met
Thr
45 50 55
Thr Val Ser IleProSer ProGlnThr TrpHisVal Phe
Gln
60 65 70
Tyr Ala Asp TyrThrCys ArgValAsp GluGluAsn Trp
Lys
75 80
3o Gln Val Glu IleProPhe GluMetVal LeuLeuAsn Pro
Asp
85 90 95
Asp Ala Glu AsnProPhe AspHisPhe GlyAlaGly Glu
Gly
100 105 110
Ser Gly Leu GluPhePhe PheLeuLeu ValLeuVal Tyr
His
115 120 125
Phe Val Thr CysIleTyr AlaGlnSer LeuTrpGln Ala
Ala
CA 02282557 1999-08-27
130 135 140
Leu Lys LysGlyGly ProMetHis MetIleLeu LysValLeu
145 150
Thr Thr AlaLeuLeu LeuGlnAla GlySerAla ValAlaAsn
5 155 160 165
Tyr Ile HisPheSer SerTyrSer LysAspGly IleGlyVal
170 175 180
Pro Phe MetGlySer LeuAlaGlu PhePheAsp IleAlaSer
185 190 195
o Gln Ile GlnMetLeu TyrLeuLeu LeuSerLeu CysMetGly
200 205 210
Trp Thr IleValArg MetLysLys SerGlnSer ArgProLeu
215 220
Gln Trp AspSerThr ProAlaSer ThrGlyIle AlaValPhe
15225 230 235
Ile Val LeuThrGln SerValLeu LeuLeuTrp GluGlnPhe
240 245 250
Glu Asp ThrGlyHis HisSerSer HisSerHis HisAsnLeu
255 260 265
2oAla Gly IleLeuLeu IleValLeu ArgIleCys LeuAlaLeu
270 275 280
Ser Leu GlyCysGly LeuTyrGln IleIleThr ValGluArg
285 290
Ser Thr LeuLysArg GluPheTyr IleThrPhe AlaLysGly
25295 300 305
Cys Ile LeuTrpPhe LeuCysHis ProSerLeu AlaCysIle
310 315 320
Ser Val IlePheAsn AspTyrGln ArgAspLys ValIleThr
325 330 335
3oIle Gly ValIleLeu GlyGlnSer ValAlaMet ValIleLeu
340 345 350
Tyr Arg LeuPheLeu SerHisSer LeuTyrTrp GluValSer
355 360
Ser Leu SerSerVal ThrLeuPro LeuThrVal SerSerGly
35365 370 375
His Lys SerArgPro HisPhe
CA 02282557 1999-08-27
61
380 385
SEQ ID N0: 3:
SEQUENCE LENGTH:
2130
SEQUENCE TYPE: nucleicacid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other acid(cDNA containing ~- and 3~-end
nucleic 5
nucleotide sequences)
io ORIGINAL SOURCE:
ORGANISM: Homo S apiens
FEATURE:
NAME/KEY: CDS
LOCATION: 97 .. 1419
IDENTIFICATION METHOD:
E
SEQUENCE DESCRIPTION: :
SEQ ID N0 3
CTCCGGCGCC CACCCCGCCT GCGGGCAGCC 50
CCCCCAGCTG CCGACGTGGG
GCCGGCGGCT GGGAGCCGAG ACGGGC 96
GCGTCGGTGC AGACCTGGAG
ATG GGG GGG CTG CGG CTG GTG GCC 126
CTG GCT
2o Met Gly Gly Leu Arg Leu Val Ala
Leu Ala
1 5 10
CTC ACG TGC TGC TGG CCG GGC AGCCAGGGT ACC 168
TGG CAG AAG
Leu Thr Cys Cys Trp Pro Gly SerGlnGly Thr
Trp Gln Lys
15 20
CTG CGG GGC AGC TTC AGC GCG GCCCAGGAC CAG 210
AGC ACC GCC
Leu Arg Gly Ser Phe Ser Ala AlaGlnAsp Gln
Ser Thr Ala
25 30 35
GGC CAG CGC ATC GGC TTC TTC CATGGTGAC GCT 252
CAC GAG CAT
Gly Gln Arg Ile Gly Phe Phe HisGlyAsp Ala
His Glu His
40 45 50
CTT CTG TGT GTC AGA AAC ATA GCAGTAGCT GGA 294
ATC AAC GTT
Leu Leu Cys Val Arg Asn Ile AlaYalAla Gly
Ile Asn Val
55 60 65
AAA GAA GCT AAA CTC CTG CAA GCCCAGGAA CTA 336
TAC TTC TGG
Lys Glu Ala Lys Leu Leu Gln AlaGlnGlu Leu
Tyr Phe Trp
70 75 80
CA 02282557 1999-08-27
62
AAG CTACAGCAA AGTCAT GGTTATAGCTGT AGTGAA 378
AGC AAA
Lys LeuGlnGln SerSerHis GlyTyrSerCys SerGluLys
85 90
TTA TCCAAAGCT CAGTTGACA ATGACCATGAAC CAGACCGAA 420
Leu SerLysAla GlnLeuThr MetThrMetAsn GlnThrGlu
95 100 105
CAT AATCTGACA GTGTCCCAG ATTCCGTCTCCA CAAACGTGG 462
His AsnLeuThr ValSerGln IleProSerPro GlnThrTrp
110 115 120
ioCAT GTGTTTTAT GCAGACAAG TATACATGCCAA GATGACAAG 504
His ValPheTyr AlaAspLys TyrThrCysGln AspAspLys
125 130 135
GAG AATTCTCAG GTGGAAGAT ATCCCATTTGAA ATGGTGTTA 546
Glu AsnSerGln ValGluAsp IleProPheGlu MetValLeu
i5 140 145 150
CTA AACCCAGAT GCCGAAGGG AATCCATTTGAT CATTTTAGT 588
Leu AsnProAsp AlaGluGly AsnProPheAsp HisPheSer
155 160
GCT GGAGAATCT GGGTTACAT GAGTTCTTTTTC CTCCTAGTC 630
2oAla GlyGluSer GlyLeuHis GluPhePhePhe LeuLeuVal
165 170 175
CTA GTGTACTTT GTGATTGCT TGCATTTATGCT CAATCATTG 672
Leu ValTyrPhe ValIleAla CysIleTyrAla GlnSerLeu
180 185 190
25TGG CAGGCTATT AAGAAAGGC GGACCCATGCAC ATGATTTTA 714
Trp GlnAlaIle LysLysGly GlyProMetHis MetIleLeu
195 200 205
AAG GTTCTGACA ACTGCATTG CTGTTACAAGCT GGTTCAGCT 756
Lys ValLeuThr ThrAlaLeu LeuLeuGlnAla GlySerAla
30 210 215 220
TTA GCTAATTAC ATTCATTTC TCCAGTTACTCC AAAGATGGA 798
Leu AlaAsnTyr IleHisPhe SerSerTyrSer LysAspGly
225 230
ATA GGGGTACCA TTTATGGGA AGTTTGGCAGAA TTTTTTGAC 840
35Ile GlyValPro PheMetGly SerLeuAlaGlu PhePheAsp
235 240 245
CA 02282557 1999-08-27
63
ATC GCT TCCCAA CAGATGTTA TACTTACTT TTGAGTCTA 882
ATT
Ile Ala SerGlnIle GlnMetLeu TyrLeuLeu LeuSerLeu
250 255 260
TGC ATG GGTTGGACA ATAGTCAGA ATGAAGAAG TCTCAAAGC 924
Cys Met GlyTrpThr IleValArg MetLysLys SerGlnSer
265 270 275
AGA CCT CTCCAGTGG GATTCTACG CCTGCATCC ACTGGCATT 966
Arg Pro LeuGlnTrp AspSerThr ProAlaSer ThrGlyIle
280 285 290
ioGCA GTA TTCATTGTC ATGACACAG AGTGTTTTG CTACTTTGG 1008
Ala Vat PheIleVal MetThrGln SerValLeu LeuLeuTrp
295 300
GAA CAG TTTGAAGAT ATCAGTCAT CATAGCTAC CATTCACAC 1050
Glu Gln PheGluAsp IleSerHis HisSerTyr HisSerHis
15305 310 315
CAC AAC TTAGCAGGG ATCCTCCTA ATTGTTCTA AGAATTTGC 1092
His Asn LeuAlaGly IleLeuLeu IleValLeu ArgIleCys
320 325 330
CTA GCA TTGTCATTA GGCTGTGGA CTCTATCAG ATCATCACA 1134
2oLeu Ala LeuSerLeu GlyCysGly LeuTyrGln IleIleThr
335 340 345
GTG GAG AGAAGTACA CTCAAAAGG GAGTTCTAC ATCACATTT 1176
Val Glu ArgSerThr LeuLysArg GluPheTyr IleThrPhe
350 355 360
25GCC AAA GGCTGTATC TTGTGGTTT TTATGCCAT CCAGTTCTT 1218
Ala Lys GlyCysIle LeuTrpPhe LeuCysHis ProValLeu
365 370
GCA TGC ATTTCTGTC ATTTTTAGC GACTACCAA AGAGACAAG 1260
Ala Cys IleSerVal IlePheSer AspTyrGln ArgAspLys
30375 ' 380 385
GTT ATT ACAATAGGT GTTATCCTT TGCCAGTCT GTTTCCATG 1302
Val Ile ThrIleGly Val-IleLeu CysGlnSer ValSerMet
390 395 400
GTT ATT CTCTACAGA CTCTTTCTG TCTCACAGT CTATACTGG 1344
35Val Ile LeuTyrArg LeuPheLeu SerHisSer LeuTyrTrp
405 410 415
CA 02282557 1999-08-27
64
GAA GTT TCT TCA CTT TCT TCA GTA ACA CTA CCA CTG ACC ATA 1386
Glu Val Ser Ser Leu Ser Ser Val Thr Leu Pro Leu Thr Ile
420 425 430
TCA TCT GGA CAC AAA AGT CGC CCT CAT TTC TGA TACTTGATTT 1429
Ser Ser Gly His Lys Ser Arg Pro His Phe
435 440
TTGTTGAGAG GAAAAGTGAA TTGGTTAAAA GAGTGCAATA AGGATCCAAA 1479
TACAGTGACT TTTTTTTCAT ACATTTAGTA TGAAAACTTG AACAGCGAAA 1529
GCAGAGCATG TTATTTATAT AACTGCATTT AAGCAGTACC AAGACTGAAA 1579
io AAAAAGGTAA TAAATGAAAT GTTTTGAAAT ATACTTAAAC AACAAACTTT 1629
GAAGAAAGTG TTGTTATAAA ATTATTGAAG CGATTTCTAT GTGGAAATAA 1679
ATGTGAAAAA TAAAACTATG ATATTTTGGT AAAATATTCA CCACTTATAA 1729
TGCCTCATCT TAATAGCTAA CTCASGTTTA ATARTCTTAT AAAAAGTAAT 1779
CAGTTAAATG AATACTTGCT TATAAATATC TAAACTAATC CACTTTATGA 1829
AATCAGTGTT ATACATTGAA TTTTAAAACT GCTGCCTTTT ATGCCTTTAA 1879
GGAAAATGTT TTTCCCTATT TTGAATTTTA AAGGAATTGA AATTCCTCCC 1929
GGAAATTAAT ATAAATAGGG TTCCCCGTTA AATGAAATAA ACCCTGGTTT 1979
AATTGGTGGG GTGGAATTAA TNCNCCCAAT TTTTTCCCGN CCCTTTTTTG 2029
GGGNCNCATT TTCCGGGTTT TAANCCTTGA ATAAACCAAA GGGTTTTTGN 2079
2o AAAAACCCTT TTTTTGAAAA AAAATTAAAA CCTTNANTTN CCTTTACCCN 2129
G 2130
SEQ ID N0: 4:
SEQUENCE LENGTH: 440
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID N0: 4
Met Gly Gly Leu Arg Leu Leu Ala Val Ala Leu Thr Cys Cys
1 5 10
Trp Trp Pro Gln Gly Ser Gln Gly Lys Thr Leu Arg Gly Ser
15 20 25
Phe Ser Ser Thr Ala Ala Gln Asp Ala Gln Gly Gln Arg Ile
30 35 40
Gly His Phe Glu Phe His Gly Asp His Ala Leu Leu Cys Val
50 55
CA 02282557 1999-08-27
Arg IleAsnAsn IleAlaValAla ValGlyLys GluAlaLys
60 65 70
Leu TyrLeuPhe GlnAlaGlnGlu TrpLeuLys LeuGlnGln
75 80
5 Ser SerHisGly TyrSerCysSer GluLysLeu SerLysAla
85 90 95
Gln LeuThrMet ThrMetAsnGln ThrGluHis AsnLeuThr
100 105 110
Val SerGlnIle ProSerProGln ThrTrpHis ValPheTyr
0 115 120 125
Ala AspLysTyr ThrCysGlnAsp AspLysGlu AsnSerGln
130 135 140
Val GluAspIle ProPheGluMet ValLeuLeu AsnProAsp
145 150
~5Ala GluGlyAsn ProPheAspHis PheSerAla GlyGluSer
155 160 165
Gly LeuHisGlu PhePhePheLeu LeuValLeu ValTyrPhe
170 175 180
Val IleAlaCys IleTyrAlaGln SerLeuTrp GlnAlaIle
20 185 190 195
Lys LysGlyGly ProMetHisMet IleLeuLys ValLeuThr
200 205 210
Thr AlaLeuLeu LeuGlnAlaGly SerAlaLeu AlaAsnTyr
215 220
25Ile HisPheSer SerTyrSerLys AspGlyIle GlyValPro
225 230 235
Phe MetGlySer LeuAlaGluPhe PheAspIle AlaSerGln
240 245 250
Ile GlnMetLeu TyrLeuLeuLeu SerLeuCys MetGlyTrp
30 255 260 265
Thr IleValArg MetLysLysSer GlnSerArg ProLeuGln
270 275 280
Trp AspSerThr ProAlaSerThr GlyIleAla ValPheIle
285 290
35Val MetThrGln SerValLeuLeu LeuTrpGlu GlnPheGlu
295 300 305
CA 02282557 1999-08-27
66
Asp Ile Ser His His Ser Tyr His Ser His His Asn Leu Ala
310 315 320
Gly Ile Leu Leu Ile Val Leu Arg Ile Cys Leu Ala Leu Ser
325 330 335
Leu Gly Cys Gly Leu Tyr Gln Ile Ile Thr Val Glu Arg Ser
340 345 350
Thr Leu Lys Arg Glu Phe Tyr Ile Thr Phe Ala Lys Gly Cys
355 360
Ile Leu Trp Phe Leu Cys His Pro Val Leu Ala Cys Ile Ser
0 365 370 375
Val Ile Phe Ser Asp Tyr Gln Arg Asp Lys Val Ile Thr Ile
380 385 390
Gly Val Ile Leu Cys Gln Ser Val Ser Met Val Ile Leu Tyr
395 400 405
Arg Leu Phe Leu Ser His Ser Leu Tyr Trp Glu Val Ser Ser
410 415 420
Leu Ser Ser Val Thr Leu Pro Leu Thr Ile Ser Ser Gly His
425 430
Lys Ser Arg Pro His Phe
435 440
SEQ ID N0: 5:
SEQUENCE LENGTH: 1918
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA containing 5~- and 3~-end
nucleotide sequences)
ORIGINAL SOURCE:
3o ORGANISM: Mus
FEATURE:
NAME/KEY: CDS
LOCATION: 82 .. 1383
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 5
GGCACGAGCC GCCCTCTGCT GCCGACGTGG GCTGCAGGCC GCAGGCGGTT 50
CA 02282557 1999-08-27
67
GCCGGGCGAG 87
CAAACGGAGC
GGGCGGCGGG
C
ATG
GGC
Met
Gly
1
GGC CTGCGGCTG CTGGCGGTA GCCCTCACG TGCAGCTGC TGG129
Gly LeuArgLeu LeuAlaVal AlaLeuThr CysSerCys Trp
5 10 15
TGG CCGCAGGGC GGCCAGGGC AAGACCCTG CGTGGCAGC TTC171
Trp ProGlnGly GlyGlnGly LysThrLeu ArgGlySer Phe
20 25 30
to AGC AGCGCCGCG GCCCGCGAC GCCCAGGGC CAGAGCATC GGC213
Ser SerAlaAla AlaArgAsp AlaGlnGly GlnSerIle Gly
35 40
CAT TTCGAGTTC CACCGAATC AACAACGTA GCAGTGGCT GTT255
His PheGluPhe HisArgIle AsnAsnVal AlaValAla Val
45 50 55
GGA AAAGAAGCT AAACTCTAC CTGTTCCAA GCCGAGGAA TGG297
Gly LysGluAla LysLeuTyr LeuPheGln AlaGlnGlu Trp
60 65 70
CTG AAGCTGCTG GAGAGCAGC CCCGGCTAC AGCTGCAGT GAG339
2o Leu LysLeuLeu GluSerSer ProGlyTyr SerCysSer Glu
75 80 85
CGG CTAGCCCGA GCTCAGCTG ACAGTGACA GTGACCCAG ACG381
Arg LeuAlaArg AlaGlnLeu ThrValThr ValThrGln Thr
90 95 100
GAG CACAACCTC ACAGTGTCC CAGCTGCCC GCTCCCCAG ACA423
Glu HisAsnLeu ThrValSer GlnLeuPro AlaProGln Thr
105 110
TGG CGAGTGTTC TATGCCGAC AAGTTCACC TGCAGGGAT GAC465
Trp ArgValPhe TyrAlaAsp LysPheThr CysArgAsp Asp
115 120 125
TCA NASAGCCCC CAGGGGGAG GAGATCCCC TTTGAAATG GTG507
Ser XxxSerPro GlnGlyGlu GluIlePro PheGluMet Val
130 135 140
CTC CTCAACCCG GACGCCGAG GGAAACCCG CTGGATCAT TTT549
Leu LeuAsnPro AspAlaGlu GlyAsnPro LeuAspHis Phe
145 150 155
CA 02282557 1999-08-27
68
AGC GCCAGAGAG TCCGGGCTCCAC GAGTTCTTT TTCCTCCTC 591
Ser AlaArgGlu SerGlyLeuHis GluPhePhe PheLeuLeu
160 165 170
GTC CTAGTGTAC TTTGTGACTGCG TGCATCTAT GCGCAGTCT 633
Val LeuValTyr PheValThrAla CysIleTyr AlaGlnSer
175 180
CTG TGGCAGGCT ATGAAGAAGGGA GGACCCATG CACACCATC 675
Leu TrpGlnAla MetLysLysGly GlyProMet HisThrIle
185 190 195
ioTTA AAGGTCCTC ACCACTGCACTG CTGCTTCAA GCTGCTTCA 717
~
Leu LysValLeu ThrThrAlaLeu LeuLeuGln AlaAlaSer
200 205 210
GCC TTAGCTAAT TACATCCACTTG TCCAGGTAC TCCAGAGAT 759
Ala LeuAlaAsn TyrIleHisLeu SerArgTyr SerArgAsp
215 220 225
GGG CTAGGAGTG CCTCTCATAGGA AGCCTGGCA GAAGTTTTT 801
Gly LeuGlyVal ProLeuIleGly SerLeuAla GluValPhe
230 235 240
GAC ATTGCCTCC CAAATTCAGATG CTGTACCTG CTTCTGAGC 843
2oAsp IleAlaSer GlnIleGlnMet LeuTyrLeu LeuLeuSer
245 250
CTG TGTATGGGC TGGACAATAGTG CGGATGAAG AAGTCGCAG 885
Leu CysMetGly TrpThrIleVal ArgMetLys LysSerGln
255 260 265
25AGC AGACCGCTC CAGTGGGACTCG ACACCCGCG TCCACGGGC 927
Ser ArgProLeu GlnTrpAspSer ThrProAla SerThrGly
270 275 280
ATC GCAGTTTTC ATYGTCATCACA CAGAGCATT TTGCTACTY 969
Ile AlaValPhe IleValIleThr GlnSerIle LeuLeuLeu
30 285 290 295
TGG GAGCAGTTT GAAGACACCAGT CACCACAGC GCACATTCA 1011
Trp GluGlnPhe GluAspThrSer HisHisSer AlaHisSer
300 305 310
CAC CGCAGCTTA GCCGGGCTCTTG CTGATTGTC TTACGGATC 1053
35His ArgSerLeu AlaGlyLeuLeu LeuIleVal LeuArgIle
315 320
CA 02282557 1999-08-27
69
TGC CTG GCG CTG TCG CTG GGC TGC GGA CAG GTC 1095
CTT TAC ATC
Cys Leu Ala Leu Ser Leu Gly Cys Gly Gln Val
Leu Tyr Ile
325 330 335
ACA GTG GAG AGG AGC GCG CTC AAG AGA TAC ATC 1137
GAG TTC ACG
Thr Val Glu Arg Ser Ala Leu Lys Arg Tyr Ile
Glu Phe Thr
340 345 350
TTT GCC AAG GGC TGC ATC CTG TGG TTC CAG CCA 1179
TTG TGC GCG
Phe Ala Lys Gly Cys Ile Leu Trp Phe Gln Pro
Leu Cys Ala
355 360 365
io CTC GCA TGC ATT GCT GTC GCT TTT AAT CAA AGA 1221
GAC TAC GAT
Leu Ala Cys Ile Ala Val Ala Phe Asn Gln Arg
Asp Tyr Asp
370 375 380
AAG CTT ATC ACA GTA GGT GTC ATC CTG GCC GTG 1263
TGT CAG GCC
Lys Leu Ile Thr Val Gly Val Ile Leu Ala Val
Cys Gln Ala
i5 385 390
ATG GTC ATT CTG TAC AGA CTT TTC CTG AGT CTT 1305
TCC CAC TAC
Met Val Ile Leu Tyr Arg Leu Phe Leu Ser Leu
Ser His Tyr
395 400 405
TGG GAG GTC TCC TCG CTC TCC TCA GTA CCA CTG 1347
ACG CTA ACC
2o Trp Glu Val Ser Ser Leu Ser Ser Val Pro Leu
Thr Leu Thr
410 415 420
ATC TCG TCT GCA CAC AGA GGG CGC CCT TGA 1383
CAT TTC
Ile Ser Ser Ala His Arg Gly Arg Pro
His Phe
425 430
25 TGCTTGAGTT TTGTGGAGAG AACCAGTGAA TGGAGAAGTGCAATAGGATC1433
CAACGCAGCA CCGTCTTGCT GTGCCTTTGC GTGACAGCTGAGCGGTGGAA1483
GCAGGGCGTC TTATTTATAG AACTGAACGT CAGCGGGCTCAGCAGAAAGG1533
AATAGAAGCT CCGGAGTGAA CTCAAACAGT GAACTTCCCAGAAAGAATGT1583
TGTTTCAAGG TGACTGAAAC AGTTTCCACG TGGAAATAAATGTGAAAAGG1633
3o ACTGCTTAGA GTACACGTGG GCCAGGTGGT CACACCTGCGATGCCTCGTC1683
ACTAGCAAAC TCAGGCCTGA TAGTCCTACA GTATTCACCTAGACAATACT1733
TGCCTGTGCG TGCCCAGCTC GCCCAGTTAT GAAATCAGCGGGATGTGCTG1783
ATTTTAAAAC TACTTCTTTT TATCCTTTAA AGAACGTGCATTTCAAATTA1833
TAATTTAAAG GACTTGAAAG TGAAATTACT TAGGAAATAAATAGAAAATA1883
35 TGTTAACAGT TAAACGAAAA AAA~~AAAAAA AAAAA 1918
CA 02282557 1999-08-27
SEQ ID N0: fi
SEQUENCE LENGTH: 433
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
5 MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID N0: 6
Met Gly Gly Leu Arg Leu Leu Ala Val Ala Leu
1 5 10
Thr Cys Ser Cys Trp Trp Pro Gln Gly Gly Gln
io 15 20
Gly Lys Thr Leu Arg Gly Ser Phe Ser Ser Ala
25 30
Ala Ala Arg Asp Ala Gln Gly Gln Ser Ile Gly
35 40
~5 His Phe Glu Phe His Arg Ile Asn Asn Val Ala Val Ala Val
45 50 55
Gly Lys Glu Ala Lys Leu Tyr Leu Phe Gln Ala Gln Glu Trp
60 65 70
Leu Lys Leu Leu Glu Ser Ser Pro Gly Tyr Ser Cys Ser Glu
20 75 80 85
Arg Leu Ala Arg Ala Gln Leu Thr Val Thr Val Thr Gln Thr
90 95 100
Glu His Asn Leu Thr Val Ser Gln Leu Pro Ala Pro Gln Thr
105 110
25 Trp Arg Val Phe Tyr Ala Asp Lys Phe Thr Cys Arg Asp Asp
115 120 125
Ser Xxx Ser Pro Gln Gly Glu Glu Ile Pro Phe Glu Met Val
130 135 140
Leu Leu Asn Pro Asp Ala Glu Gly Asn Pro Leu Asp His Phe
30 145 150 155
Ser Ala Arg Glu Ser Gly Leu His Glu Phe Phe Phe Leu Leu
160 165 170
Val Leu Val Tyr Phe Val Thr Ala Cys Ile Tyr Ala Gln Ser
175 180
35 Leu Trp Gln Ala Met Lys Lys Gly Gly Pro Met His Thr Ile
185 190 195
CA 02282557 1999-08-27
71
Leu LysVal LeuThrThr AlaLeuLeu LeuGlnAla AlaSer
200 205 210
Ala LeuAla AsnTyrIle HisLeuSer ArgTyrSer ArgAsp
215 220 225
Gly LeuGly ValProLeu IleGlySer LeuAlaGlu ValPhe
230 235 240
Asp IleAla SerGlnIle GlnMetLeu TyrLeuLeu LeuSer
245 250
Leu CysMet GlyTrpThr IleValArg MetLysLys SerGln
l0 255 260 265
Ser ArgPro LeuGlnTrp AspSerThr ProAlaSer ThrGly
270 275 280
Ile AlaVal PheIleVal IleThrGln SerIleLeu LeuLeu
285 290 295
Trp GluGln PheGluAsp ThrSerHis HisSerAla HisSer
'
300 305 310
His ArgSer LeuAlaGly LeuLeuLeu IleValLeu ArgIle
315 320
Cys LeuAla LeuSerLeu GlyCysGly LeuTyrGln ValIle
325 330 335
Thr ValGlu ArgSerAla LeuLysArg GluPheTyr IleThr
340 345 350
Phe AlaLys GlyCysIle LeuTrpPhe LeuCysGln ProAla
355 360 365
Leu AlaCys IleAlaVal AlaPheAsn AspTyrGln ArgAsp
370 375 380
Lys LeuIle ThrValGly ValIleLeu CysGlnAla ValAla
385 390
Met ValIle LeuTyrArg LeuPheLeu SerHisSer LeuTyr
395 400 405
Trp GluVal SerSerLeu SerSerVal ThrLeuPro LeuThr
410 415 420
Ile SerSer AlaHisArg GlyArgPro HisPhe
425 430
SEQ ID N0: 7:
CA 02282557 1999-08-27
72
SEQUENCE LENGTH: 2857
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA containing 5~- and 3~-end
nucleotide sequences)
ORIGINAL SOURCE:
ORGANISM: Oryctolagus
FEATURE:
to NAME/KEY: CDS
LOCATION: 415 .. 2199
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 7
GTGTTACTGT GTTTCACTAA ATGTTTGAAG GCTGTCGGACTTTTTGAATC50
ATATGATCTC CTGAAAGTAG TTCACATTGT TCAGTTCGTTTTTATATTAA100
AACTTGGGAC TGCATTTTTT ATGGTTTTGT TTCAAAAGCCATTTTCTTCT150
GGGAAAACTA TTACCAAACA CCAGTGGATC ACAATATTTAAACATGCAGT200
TGCCGGGTGT ATCATTTCAC TCTTGTGGTT TTTTGGCCTTACCCTTTGTG250
GACCACTAAG GACTTTGCTG CTGTTTGAAC ACAGTGAAATTGTTGTCATC300
2o TCGCTCCTCA GTGTTTTGTT CACCAGTTCT GGAGGAGGACCAGCAAAGAC350
AAGAGGGGCT GCTTTTTTCA TCATTGCTGT GATCTGTTTATTGCTTTTTG400
ACAATGATGA TCTC 414
ATG GCT AAA ATG GCA GAA CAC CCT GAA CAT GAC 456
GGA CAT AGT
Met Ala Lys Met Ala Glu His Pro Glu His Asp
Gly His Ser
1 5 10
GCT CTA ACT CAC ATG CTT TAC ACA GCC TTC TTA 498
ATT GCC GGT
Ala Leu Thr His Met Leu Tyr Thr Ala Phe Leu
Ile Ala Gly
15 ZO 25
GTG GCA GAT CAC AAG GGT GGA GTA TTG GTA CTG 540
TTG CTA GCT
3o Val Ala Asp His Lys Gly Gly Val Leu Val Leu
Leu Leu Ala
35 40
TTG TGT TGT AAA GTT GGT TTT CAC ACA AGA AAA 582
GCT TCC CTC
Leu Cys Cys Lys Val Gly Phe His Thr Arg Lys
Ala Ser Leu
45 50 55
TCT ATA GAT GTT GGG GGA GCC AAA CGT GCT TTA 624
CTT CAA TCC
Ser Ile Asp Val Gly Gly Ala Lys Arg Ala Leu
Leu Gln Ser
CA 02282557 1999-08-27
73
60 65 70
CAT CTTGTTTCT GTGCTTCTC TTGTGCCCA TGGGTCATTGTT 666
His LeuValSer ValLeuLeu LeuCysPro TrpValIleVal
75 80
CTT TCTATGACA ACTGAGAGT AAAGTTGAG TCTTGGTTTTCT 708
Leu SerMetThr ThrGluSer LysValGlu SerTrpPheSer
85 90 95
CTC ATTATGCCT TTCACGATG GTTATTTTT TTTGTCWTGATC 750
Leu IleMetPro PheThrMet ValIlePhe PheValXaaIle
io 100 105 110
CTG GATTTCTAC GTGGATTCC ATTTGTTCA GTCAAAATGGAA 792
Leu AspPheTyr ValAspSer IleCysSer ValLysMetGlu
115 120 125
GTT TCCAAATGT GCCCGCTAT GGATCCTTG CCCATTTTTATT 834
~5 Val SerLysCys AlaArgTyr GlySerLeu ProIlePheIle
130 135 140
AGT GCTCTCCTT TTTGGAAAT TTCTGGACC CACCCCATAACT 876
Ser AlaLeuLeu PheGlyAsn PheTrpThr HisProIleThr
145 150
2o GAC CAGCTTCGG GCAATGAGC AGAGCAGCA CACCAGGGGAGC 918
Asp GlnLeuArg AlaMetSer ArgAlaAla HisGlnGlySer
155 160 165
ACG GAACACGTT CTGTCTGGA GGAGTGGTC GTGAGCGCAGTG 960
Thr GluHisVal LeuSerGly GlyValVal ValSerAlaVal
25 170 175 180
TTC TTCATCTTG TCTGCCAAC ATCCTGTCA TCTCCTTCGAAG 1002
Phe PheIleLeu SerAlaAsn IleLeuSer SerProSerLys
185 190 195
AGG GGGCAGAAG GGCACCCTG ATTGGATAC TCTCCTGAAGGA 1044
3o Arg GlyGlnLys GlyThrLeu IleGlyTyr SerProGluGly
200 205 210
GCA CCTCTTTAC AACTTCATG GGGGATGCT TTTCAGCACAGC 1086
Ala ProLeuTyr AsnPheMet GlyAspAla PheGlnHisSer
215 220
35 TCA CAGTCCGTG CCTCGGTTT ATTAAGGAA TCGCTGAAACAG 1128
Ser GlnSerVal ProArgPhe IleLysGlu SerLeuLysGln
CA 02282557 1999-08-27
74
225 230 235
ATT CTTGAGGAG AGTGACTCTAGG CAGATCTTT TACTTCTTG 1170
Ile LeuGluGlu SerAspSerArg GlnIlePhe TyrPheLeu
240 245 250
TGC TTGAATCTG CTTTTTACCTTT GTGGAATTA TTCTATGGA 1212
Cys LeuAsnLeu LeuPheThrPhe ValGluLeu PheTyrGly
255 260 265
GTG CTGACGAAT AGTCTGGGTCTG ATCTCAGAT GGCTTTCAC 1254
Val LeuThrAsn SerLeuGlyLeu IleSerAsp GlyPheHis
io 270 275 280
ATG CTCTTTGAC TGCTCTGCCTTG GTCATGGGA CTTTTTGCT 1296
Met LeuPheAsp CysSerAlaLeu ValMetGly LeuPheAla
285 290
GCC CTGATGAGT AGATGGAAAGCA ACTCGGATT TTCTCCTAC 1338
Ala LeuMetSer ArgTrpLysAla ThrArgIle PheSerTyr
295 300 305
GGG TATGGCCGA ATAGAAATTCTT TCTGGATTT ATTAATGGA 1380
Gly TyrGlyArg IleGluIleLeu SerGlyPhe IleAsnGly
310 315 320
2o CTT TTTCTAATA GTAATAGCTTTT TTTGTGTTT ATGGAGTCA 1422
Leu PheLeuIle ValIleAlaPhe PheValPhe MetGluSer
325 330 335
GTT GCCAGATTG ATTGATCCTCCG GAATTAGAC ACAAACATG 1464
Val AlaArgLeu IleAspProPro GluLeuAsp ThrAsnMet
340 345 350
CTA ACACCAGTG TCAGTTGGAGGG CTGATAGTA AACCTTATT 1506
Leu ThrProVal SerValGlyGly LeuIleVal AsnLeuIle
355 360
GGT ATCTGTGCC TTTAGCCACGCC CATAATCAC ACCCATGGA 1548
3o Gly IleCysAla PheSerHisAla HisAsnHis ThrHisGly
365 370 375
TCT TCCCAAGGA AGCTGTCACTCA TCCGATCAC AGCCATTCA 1590
Ser SerGlnGly SerCysHisSer SerAspHis SerHisSer
380 385 390
CAC CACATGCAT GGACACAGTGAC CATGGACAT GGTCACAGC 1632
His HisMetHis GlyHisSerAsp HisGlyHis GlyHisSer
CA 02282557 1999-08-27
395 400 405
CAT GGA TCCCCAGGC GGCGGCATG AATGCTAAC ATGAGGGGT 1674
His Gly SerProGly GlyGlyMet AsnAlaAsn MetArgGly
410 415 420
5 GTG TTT TTCCATGTT TTGGCAGAC ACGCTTGGC AGTATTGGT 1716
Val Phe PheHisVal LeuAlaAsp ThrLeuGly SerIleGly
425 430
GTG ATT GTATTTACA GTTTTTATA GAGCAGTTT GGGTGGTTC 1758
Val Ile ValPheThr ValPheIle GluGlnPhe GlyTrpPhe
io 435 440 445
ATT GCG GATCCCCTC TGTTCTCTC TTTATTGCT GTATTAATA 1800
Ile Ala AspProLeu CysSerLeu PheIleAla ValLeuIle
450 455 460
TTT CTC AGTGTTGTC CCACTGATC AAAGATGCC TGTCAGGTT 1842
~5 Phe Leu SerValVal ProLeuIle LysAspAla CysGlnVal
465 470 475
CTA CTT TTGAGACTG CCACCAGAG TATGAAAAA GAACTACAT 1884
Leu Leu LeuArgLeu ProProGlu TyrGluLys GluLeuHis
480 485 490
2o ATT GCT TTAGAAAAG ATACAAAAA ATTGARGGA TTAATATCA 1926
Ile Ala LeuGluLys IleGlnLys IleGluGly LeuIleSer
495 500
TAC CGA GATCCTCAT TTCTGGCGC CATTCTGCC AGTATTGTG 1968
Tyr Arg AspProHis PheTrpArg HisSerAla SerIleVal
25 505 510 515
GCA GGA ACAATTCAT ATACAAGTG ACATCTGAT GTGCTAGAA 2010
Ala Gly ThrIleHis IleGlnVal ThrSerAsp ValLeuGlu
520 525 530
CAA AGA ATAGTACAG CAGGTTACA GGAATACTT AAAGATGCA 2052
3o Gln Arg IleValGln GlnValThr GlyIleLeu LysAspAla
535 540 545
GGA GTA AACAATTTA ACAATTCAA GTAGAAAAA GAAGCATAC 2094
Gly Val AsnAsnLeu ThrIleGln ValGluLys GluAlaTyr
550 555 560
35 TTT CAA CATATGTCT GGCCTAAGT ACTGGATTT CATGATGTT 2136
Phe Gln HisMetSer GlyLeuSer ThrGlyPhe HisAspVal
CA 02282557 1999-08-27
76
565 570
CTG GCT ATG ACA AAA CAA ATG GAG TCC ATG AAA TAC TGC AAG 2178
Leu Ala Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys
575 580 585
GAT GGC ACT TAC ATT ATG TGA GAGAACTCAC AGATTACCCC 2219
Asp Gly Thr Tyr Ile Met
590
CGATGTGAGC AGTGAAGATT CAGTGACTCA GTGTTGTAAC ATTGCCAGCA 2269
GGACAGAAAC TGCGTGTAAT TGTACAGAGA TTTTAAAGCT CCCTATTCTT 2319
to GGATCAAGGA CTCTTTCCTA AAGGAAATTT AAATATTGAT TGAAACATTG 2369
ATCACACAGT AAAATAGTGA TTTGAGTTAT GTATTTTAAA TGACTCTTAC 2419
AATTTGAACA TAATGTGTCT CATCATCTTC AGAAATGGAC ACAATGATGG 2469
ATTCTAATGA AGACCAAAAG TACTTCTGTG TTTCCTTTCT GTCAGAAAGC 2519
ATCTCCATTG TAAATATGTA TTTACATGTT TATTACAAAG ATCCAAATGA 2569
~5 AAAATTTTTA GTCCATTTTT TGCATAGCCT AAAGATAAAA TAGGAATAAA 2619
AGTTCTATAT TTATGAATTT TCTGTACATA AAACTGGTTT CTAATTATAA 2669
CTGAAGTCCA CTGGGTAAAA TCTGTATTGC CACCTTAAAT GTAAACTAAA 2719
TTATTTGAGA GAAACTTCAA CCACTGATAT GACATAAGCA GTGAGAACAG 2769
GGAGTCTATA ACATTACAGT TTTGGATGTT ACCAAAACCA ACCACTCTGT 2819
2o AAAATAAATT TTTTACTTTT GTCAAAAAAA AAAAAAAA 2857
SEQ ID N0: 8:
SEQUENCE LENGTH: 594
SEQUENCE TYPE: amino acid
25 TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID N0: 8
Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser
1 5 10
3o Ala Leu Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly
20 25
Val Ala Asp His Lys Gly Gly Val Leu Leu Leu Val Leu Ala
30 35 40
Leu Cys Cys Lys Val Gly Phe His Thr Ala Ser Arg Lys Leu
35 45 50 55
Ser Ile Asp Val Gly Gly Ala Lys Arg Leu Gln Ala Leu Ser
CA 02282557 1999-08-27
77
60 65 70
His Leu ValSerVal LeuLeuLeu CysProTrp ValIleVal
75 80
Leu Ser MetThrThr GluSerLys ValGluSer TrpPheSer
85 90 95
Leu Ile MetProPhe ThrMetVal IlePhePhe ValXaaIle
100 105 110
Leu Asp PheTyrVal AspSerIle CysSerVal LysMetGlu
115 120 125
o Val Ser LysCysAla ArgTyrGly SerLeuPro IlePheIle
130 135 140
Ser Ala LeuLeuPhe GlyAsnPhe TrpThrHis ProIleThr
145 150
Asp Gln LeuArgAla MetSerArg AlaAlaHis GlnGlySer
i5 155 160 165
Thr Glu HisValLeu SerGlyGly ValValVal SerAlaVal
170 175 180
Phe Phe IleLeuSer AlaAsnIle LeuSerSer ProSerLys
185 190 195
2o Arg Gly GlnLysGly ThrLeuIle GlyTyrSer ProGluGly
200 205 210
Ala Pro LeuTyrAsn PheMetGly AspAlaPhe GlnHisSer
215 220
Ser Gln SerValPro ArgPheIle LysGluSer LeuLysGln
25 225 230 235
Ile Leu GluGluSer AspSerArg GlnIlePhe TyrPheLeu
240 245 250
Cys Leu AsnLeuLeu PheThrPhe ValGluLeu PheTyrGly
255 260 265
3o Val Leu ThrAsnSer LeuGlyLeu IleSerAsp GlyPheHis
270 275 280
Met Leu PheAspCys SerAlaLeu ValMetGly LeuPheAla
285 290
Ala Leu MetSerArg TrpLysAla ThrArgIle PheSerTyr
35 295 300 305
Gly Tyr GlyArgIle GluIleLeu SerGlyPhe IleAsnGly
CA 02282557 1999-08-27
78
310 315 320
Leu PheLeuIle ValIleAla PhePheValPhe MetGluSer
325 330 335
Val AlaArgLeu IleAspPro ProGluLeuAsp ThrAsnMet
340 345 350
Leu ThrProVal SerValGly GlyLeuIleVal AsnLeuIle
355 360
Gly IleCysAla PheSerHis AlaHisAsnHis ThrHisGly
365 370 375
io Ser SerGlnGly SerCysHis SerSerAspHis SerHisSer
380 385 390
His HisMetHis GlyHisSer AspHisGlyHis GlyHisSer
395 400 405
His GlySerPro GlyGlyGly MetAsnAlaAsn MetArgGly
410 415 420
Val PhePheHis ValLeuAla AspThrLeuGly SerIleGly
425 430
Val IleValPhe ThrValPhe IleGluGlnPhe GlyTrpPhe
435 440 445
2o Ile AlaAspPro LeuCysSer LeuPheIleAla YalLeuIle
450 455 460
Phe LeuSerVal ValProLeu IleLysAspAla CysGlnVal
465 470 475
Leu LeuLeuArg LeuProPro GluTyrGluLys GluLeuHis
480 485 490
Ile AlaLeuGlu LysIleGln LysIleGluGly LeuIleSer
495 500
Tyr ArgAspPro HisPheTrp ArgHisSerAla SerIleVal
505 510 515
3o Ala GlyThrIle HisIleGln ValThrSerAsp ValLeuGlu
520 525 530
Gln ArgIleVal GlnGlnVal ThrGlyIleLeu LysAspAla
535 540 545
Gly ValAsnAsn LeuThrIle GlnValGluLys GluAlaTyr
550 555 560
Phe GlnHisMet SerGlyLeu SerThrGlyPhe HisAspVal
CA 02282557 1999-08-27
79
565 570
Leu Ala Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys
575 580 585
Asp Gly Thr Tyr Ile Met
590
SEQ ID N0: 9:
SEQUENCE LENGTH: 2519
SEQUENCE TYPE: nucleic acid
to STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA containing 3~-end nucleotide
sequence)
ORIGINAL SOURCE:
ORGANISM: Homo sapiens
FEATURE:
NAME/KEY: CDS
LOCATION: 1 .. 1785
IDENTIFICATION METHOD: E
2o SEQUENCE DESCRIPTION: SEQ ID N0: 9
ATG GCT AAA ATG GCT GAA CAC CCT.GAA GGA CAT CAT GAC AGT 42
Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser
1 5 10
GCT CTA ACT CAT ATG CTT TAC ACA GCC ATT GCC TTC TTA GGT 84
Ala Leu Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly
15 20 25
GTG GCA GAT CAC AAG GGT GGA GTA TTA TTG CTA GTA CTG GCT 126
Val Ala Asp His Lys Gly Gly Yal Leu Leu Leu Val Leu Ala
35 40
3o TTG TGT TGT AAA GTT GGT TTT CAT ACA GCT TCC AGA AAG CTC 168
Leu Cys Cys Lys Val Gly Phe His Thr Ala Ser Arg Lys Leu
45 50 55
TCT GTC GAC GTT GGT GGA GCT AAA CGT CTT CAA GCT TTA TCT 210
Ser Val Asp Val Gly Gly Ala Lys Arg Leu Gln Ala Leu Ser
60 65 70
CAT CTT GTT TCT GTG CTT CTC TTG TGC CCA TGG GTC ATT GTT 252
CA 02282557 1999-08-27
His LeuValSer ValLeuLeuLeu CysProTrp ValIleVal
75 80
CTT TCTGTGACA ACTGAGAGTAAA GTGGAGTCT TGGYTTTCT 294
Leu SerValThr ThrGluSerLys ValGluSer TrpXaaSer
5 85 90 95
CTC ATTATGCCT TTTGCAACGGTT ATCTTTTTT GTCATGATC 336
Leu IleMetPro PheAlaThrVal IlePhePhe ValMetIle
100 105 110
CTG GATTTCTAC GTGGATTCCATT TGTTCAGTC AAAATGGAA 378
io Leu AspPheTyr ValAspSerIle CysSerVal LysMetGlu
115 120 125
GTT TCCAAATGT GCTCGTTATGGA TCCTTTCCC ATTTTTATT 420
Val SerLysCys AlaArgTyrGly SerPhePro IlePheIle
130 135 140
~5 AGT GCTCTCCTT TTTGGAAATTTT TGGACACAT CCAATAACA 462
Ser AlaLeuLeu PheGlyAsnPhe TrpThrHis ProIleThr
145 150
GAC CAGCTTCGG GCTATGAACAAA GCAGCACAC CAGGAGAGC 504
Asp GlnLeuArg AlaMetAsnLys AlaAlaHis GlnGluSer
20 155 160 165
ACT GAACACGTC CTGTCTGGAGGA GTGGTAGTG AGTGCTATA 546
Thr GluHisVal LeuSerGlyGly ValValVal SerAlaIle
170 175 180
TTC TTCATTTTG TCTGCCAATATC TTATCATCT CCCTCTAAG 588
25 Phe PheIleLeu SerAlaAsnIle LeuSerSer ProSerLys
185 190 195
AGA GGACAAAAA GGTACCCTTATT GGATATTCT CCTGAAGGA 630
Arg GlyGlnLys GlyThrLeuIle GlyTyrSer ProGluGly
200 205 210
3o ACA CCTCTTTAT AACTTCATGGGT GATGCTTTT CAGCATAGC 672
Thr ProLeuTyr AsnPheMetGly AspAlaPhe GlnHisSer
215 220
TCT CAATCGATC CCTAGGTTTATT AAGGAATCA CTAAAACAA 714
Ser GlnSerIle ProArgPheIle LysGluSer LeuLysGln
35 225 230 235
ATT CTTGAGGAG AGTGACTCTAGG CAGATCTTT TACTTCTTG 756
CA 02282557 1999-08-27
81
Ile Leu GluGluSer AspSerArg GlnIlePheTyr PheLeu
240 245 250
TGC TTG AATCTGCTT TTTACCTTT GTGGAATTATTC TATGGC 798
Cys Leu AsnLeuLeu PheThrPhe ValGluLeuPhe TyrGly
255 260 265
GTG CTG ACCAATAGT CTGGGCCTG ATCTCGGATGGA TTCCAC 840
Val Leu ThrAsnSer LeuGlyLeu IleSerAspGly PheHis
270 275 280
ATG CTT TTTGACTGC TCTGCTTTA GTCATGGGACTT TTTGCT 882
to Met Leu PheAspCys SerAlaLeu ValMetGlyLeu PheAla
285 290
GCC CTG ATGAGTAGG TGGAAAGCC AGTCGGATTTTC TCCTAT 924
Ala Leu MetSerArg TrpLysAla ThrArgIlePhe SerTyr
295 300 305
~5 GGG TAC GGCCGAATA GAAATTCTG TCTGGATTTATT AATGGA 966
Gly Tyr GlyArgIle GluIleLeu SerGlyPheIle AsnGly
310 315 320
CTT TTT CTAATAGTA ATAGCGTTT TTTGTGTTTATG GAGTCA 1008
Leu Phe LeuIleVal IleAlaPhe PheValPheMet GluSer
20 325 330 335
GTG GCT ARATTGATT GATCCTCCA GAATTAGACACT CACATG 1050
Val Ala XaaLeuIle AspProPro GluLeuAspThr HisMet
340 345 350
TTA ACA CCAGTYTCA GTTGGAGGG CTGATAGTAAAC CTTATT 1092
25 Leu Thr ProValSer ValGlyGly LeuIleValAsn LeuIle
355 360
GGT ATC TGTGCCTTT AGCCATGCC CATAGCCATGCC CATGGA 1134
Gly Ile CysAlaPhe SerHisAla HisSerHisAla HisGly
365 370 375
3o GCT TCT CAAGGAAGC TGTCACTCA TCTGATCACAGC CATTCA 1176
Ala Ser GlnGlySer CysHisSer SerAspHisSer HisSer
380 385 390
CAY CAT ATGCATGGA CACAGTGAC CATGGGCATGGT CACAGC 1218
His His MetHisGly HisSerAsp HisGlyHisGly HisSer
35 395 400 405
CAC GGA TCTGCGGGT GGAGGCATG AATGCTAACATG AGGGGT 1260
CA 02282557 1999-08-27
82
His Gly SerAlaGly GlyGlyMet AsnAlaAsn MetArgGly
410 415 420
GTA TTT YTACATGTT TTGGCAGAT ACWCTTGGC AGCATTGGT 1302
Val Phe LeuHisVal LeuAlaAsp ThrLeuGly SerIleGly
425 430
GTG ATT GTATCCACA GTTTTTATA GAGCAGTTT GGATGGTTC 1344
Val Ile ValSerThr ValPheIle GluGlnPhe GlyTrpPhe
435 440 445
ATC GCT GACCCACTC TGTTCTCTT TTTATTGCT ATATTAATA 1386
o Ile Ala AspProLeu CysSerLeu PheIleAla IleLeuIle
450 455 460
TTT CTC AGTGTTGTT CCACTGATT AAAGATGCC TGCCAGGTT 1428
Phe Leu SerValVal ProLeuIle LysAspAla CysGlnVal
465 470 475
TTA CTC CTGAGATTG CCACCAGAA TATGGAAAA GAACTACAT 1470
Leu Leu LeuArgLeu ProProGlu TyrGlyLys GluLeuHis
480 485 490
ATT GCT TTAGAAAAG ATACAGNAA ATTGAAGGA TTAATATCA 1512
Ile Ala Le~uGluLys IleGlnXaa IleGluGly LeuIleSer
495 500
TAC CGA GACCCTCAT TTTTGGCGT CATTYTGCT AGTATTGTG 1554
Tyr Arg AspProHis PheTrpArg HisXaaAla SerIleVal
505 510 515
GCA GGA ACAATTCAT ATACAGGTG ACATYTGAT GTGCTAGAA 1596
Ala Gly ThrIleHis IleGlnVal TheXaaAsp ValLeuGlu
520 525 530
CAA AGA ATAGTACRG CAGGTTACA GGAATACTT AAAGATGCT 1638
Gln Arg IleValXaa GlnValThr GlyIleLeu LysAspAla
535 540 545
3o GGA GTA AACAATTTA ACAATTCAA GTGGAAAAG GAGGCATAC 1680
Gly Val AsnAsnLeu ThrIleGln ValGluLys GluAlaTyr
550 555 560
TTT CAA CATATGTYT GGCCTAAGT ACTGGATTT CATGATGTT 1722
Phe Gln HisMetXaa GlyLeuSer ThrGlyPhe HisAspVal
565 570
CTG GCT ATGACAAAA CAAATGGAA TCCATGAAA TACTGCAAA 1764
CA 02282557 1999-08-27
83
Leu Ala Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys
575 580 585
GAT GGT ACT TAC ATC ATG TGA GATAACTCAA GAATTACCCC 1805
Asp Gly Thr Tyr Ile Met
590
TGGAGAATAA ACAATGAAGA TTAAATGACT CAGTATTTGT AATATTGCCA 1855
GAAGGATAAA AATTACACAT TAACTGTACA GAAACAGAGT TCCCTACTAC 1905
TGGATCAAGG AATCTTTCTT GAAGGAAATT TAAATACAGA ATGAAACATT ~ 1955
AATGGTAAAA GTGGAGTAAT TATTTAAATT ATGTGTATAA AAGGAATCAA 2005
to ATTTTGAGTA AACATGATGT ATTACATCAT CTTCAAAAAT AGATATGATG 2055
GATTCTAGTG AAGACCAAAA TTACTTCTGT TTACTTTCTA TCAGGAAGCA 2105
TCTCCATTGT AAATATGTAT TTACATGTTT ATTACAAAGA CCCAAATGAA 2155
AAATTTTTAG TCCATTTTTT GCATAGCCTA AAGATAAAAT AGGAATAAAA 2205
GTTCTATATT TATGGGATTT TCTGTATATA AAACTGGTTT CTAATTATAA 2255
~5 CTTAAGTCCA TTAAGTAAAA TCTGTATTGC CACTTTAAAT GTAAACTAAA 2305
TTATTTGGGA GAAACTTCAA CCACTGATAT GAGATAAGCA ATGAGAATAG 2355
GGAAGTGTAT AACATCACAG TTTTTGATGT ATTACAAAAA TCAACCACTT 2405
TATAAAATAA ATTTTTTTTA CTTTTGGTAA AA~~AAAAAAA AA~~AAAAAAA 2455
AA~AAAAAAAA AA~~AAAAAAG CGGCCGCTGA ATTCTAGNTA GAATTCAGCG 2505
2o GCCGCTGAAT NCTA 2519
SEQ ID NO: 10:
SEQUENCE LENGTH: 594
SEQUENCE TYPE: amino acid
25 TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID NO: 10
Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser
1 5 10
3o Ala Leu Thr His Met Leu Tyr Thr Ala Ile Ala Phe Leu Gly
20 25
Val Ala Asp His Lys Gly Gly Va1 Leu Leu Leu Val Leu Ala
30 35 40
Leu Cys Cys Lys Val Gly Phe His Thr Ala Ser Arg Lys Leu
35 45 50 55
Ser Val Asp Val Gly Gly Ala Lys Arg Leu Gln Ala Leu Ser
CA 02282557 1999-08-27
84
60 65 70
His LeuValSer ValLeuLeuLeu CysProTrp ValIleVal
75 80
Leu SerValThr ThrGluSerLys ValGluSer TrpXaaSer
s 85 90 95
Leu IleMetPro PheAlaThrVal IlePhePhe ValMetIle
100 105 110
Leu AspPheTyr ValAspSerIle CysSerVal LysMetGlu
115 120 125
toVal SerLysCys AlaArgTyrGly SerPhePro IlePheIle
130 135 140
Ser AlaLeuLeu PheGlyAsnPhe TrpThrHis ProIleThr
145 150
Asp GlnLeuArg AlaMetAsnLys AlaAlaHis GlnGluSer
i5155 160 165
Thr GluHisVal LeuSerGlyGly ValValVal SerAlaIle
170 175 180
Phe PheIleLeu SerAlaAsnIle LeuSerSer ProSerLys
185 190 195
2oArg GlyGlnLys GlyThrLeuIle GlyTyrSer ProGluGly
200 205 210
Thr ProLeuTyr AsnPheMetGly AspAlaPhe GlnHisSer
215 220
Ser GlnSerIle ProArgPheIle LysGluSer LeuLysGln
25225 230 235
Ile LeuGluGlu SerAspSerArg GlnIlePhe TyrPheLeu
240 245 250
Cys LeuAsnLeu LeuPheThrPhe ValGluLeu PheTyrGly
255 260 265
3oVal LeuThrAsn SerLeuGlyLeu IleSerAsp GlyPheHis
270 275 280
Met LeuPheAsp CysSerAlaLeu ValMetGly LeuPheAla
285 290
Ala LeuMetSer ArgTrpLysAla ThrArgIle PheSerTyr
35295 300 305
Gly Tyr.GlyArg IleGluIleLeu SerGlyPhe IleAsnGly
CA 02282557 1999-08-27
310 315 320
Leu PheLeu IleValIle AlaPhePhe ValPheMet GluSer
325 330 335
Val AlaXaa LeuIleAsp ProProGlu LeuAspThr HisMet
5 340 345 350
Leu ThrPro ValSerVal GlyGlyLeu IleValAsn LeuIle
355 360
Gly IleCys AlaPheSer HisAlaHis SerHisAla HisGly
365 370 375
to Ala SerGln GlySerCys HisSerSer AspHisSer HisSer
380 385 390
His HisMet HisGlyHis SerAspHis GlyHisGly HisSer
395 400 405
His GlySer AlaGlyGly GlyMetAsn AlaAsnMet ArgGly
i5 410 415 420
Val PheLeu HisValLeu AlaAspThr LeuGlySer IleGly
425 430
Val IleVal SerThrVal PheIleGlu GlnPheGly TrpPhe
435 440 445
2o Ile AlaAsp ProLeuCys SerLeuPhe IleAlaIle LeuIle
450 455 460
Phe LeuSer ValValPro LeuIleLys AspAlaCys GlnVal
465 470 475
Leu LeuLeu ArgLeuPro ProGluTyr GlyLysGlu LeuHis
25 480 485 490
Ile AlaLeu GluLysIle GlnXaaIle GluGlyLeu IleSer
495 500
Tyr ArgAsp ProHisPhe TrpArgHis XaaAlaSer IleVal
505 510 515
3o Ala GlyThr IleHisIle GlnValThe XaaAspVal LeuGlu
520 525 530
Gln ArgIle ValXaaGln ValThrGly IleLeuLys AspAla
535 540 545
Gly ValAsn AsnLeuThr IleGlnVal GluLysGlu AlaTyr
35 550 555 560
Phe GlnHis MetXaaGly LeuSerThr GlyPheHis AspVal
CA 02282557 1999-08-27
86
565 570
Leu Ala Met Thr Lys Gln Met Glu Ser Met Lys Tyr Cys Lys
575 580 585
Asp Gly Thr Tyr Ile Met
590
SEQ ID N0: 11:
SEQUENCE LENGTH: 178
SEQUENCE TYPE: nucleic acid
~o STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA fragment)
ORIGINAL SOURCE:
ORGANISM: Oryctolagus
SEQUENCE DESCRIPTION: SEQ ID N0: 11
GTTTTTTTTT TTTTTCATAC ATTTGGTATG AAACATCGAA TAGCAAAAGC 50
AGANCATGTT TCTGTATNAC TGCATTTAAG CAGTACCAAA ACTGAANAAA 100
GGTAATAACT GAAATGTTTT AAAATACATG TAAACAATAA ACTTTCAGGA 150
AATTCTGTTG TTAAAAAAAA AAAAAAAC 178
SEQ ID N0: 12:
SEQUENCE LENGTH: 167
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA fragment)
ORIGINAL SOURCE:
ORGANISM: Oryctolagus
SEQUENCE DESCRIPTION: SEQ ID N0: 12
3o CTTGATTGCC ACCTTAAATG TAAACTAAAT TATTTGAGAG AAACTTCAAC 50
CACTGATATG ACATAAGCAG TGAGAACAGG GAGTCTATAA CATTACAGTT 100
TTGGATGTTA CCAAAACCAA CCACTCTGTA AAATAAATTT TTTACTTTTG 150
TAAAAAAAAA AAAAAAC 167
SEQ ID N0: 13:
SEQUENCE LENGTH: 24
CA 02282557 1999-08-27
87
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..24
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 13
io TCATACATTT GGTATGAAAC ATCG 24
SEQ ID N0: 14:
SEQUENCE LENGTH: 24
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
2o LOCATION: 1..24
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 14
TTTTTTTAAC AACAGAATTT CCTG 24
SEQ ID N0: 15:
SEQUENCE LENGTH: 27
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
3o MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..27
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 15
AATTTCCTGA AAGTTTATTG TTTACAT 27
CA 02282557 1999-08-27
88
SEQ ID N0: 16:
SEQUENCE LENGTH: 25
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
o LOCATION: 1..25
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 16
GAAACATGCT CTGCTTTTGC TATTC 25
SEQ ID N0: 17:
SEQUENCE LENGTH: 27
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
2o MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..27
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 17
ATCCCTGCTA AGTTGTGGTG TGAATGG 27
SEQ ID N0: 18:
SEQUENCE LENGTH: 27
3o SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NEME/KEY: primer bind
LOCATION: 1..27
CA 02282557 1999-08-27
89
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 18
TCTGCTTTGA GACTTCTTCA TCCTGAC 27
SEQ ID N0: 19:
SEQUENCE LENGTH: 27
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
to MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..27
IDENTIFICATION METHOD: E
i5 SEQUENCE DESCRIPTION: SEQ ID N0: 19
CCATCCTAAT ACGACTCACT ATAGGGC 27
SEQ ID NO: 20:
SEQUENCE LENGTH: 23
2o SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
25 NAME/KEY: primer bind
LOCATION: 1..23
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 20
ACTCACTATA GGGCTCGAGC GGC 23
SEQ ID N0: 21:
SEQUENCE LENGTH: 22
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
CA 02282557 1999-08-27
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..22
IDENTIFICATION METHOD: E
5 SEQUENCE DESCRIPTION: SEQ ID N0: 21
TTGATTGCCA CCTTAAATGT AA 22
SEQ ID N0: 22:
SEQUENCE LENGTH: 22
io SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
i5 NAME/KEY: primer bind
LOCATION: 1..22
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 22
GTGGTTGGTT TTGGTAACAT CC 22
SEQ ID N0: 23:
SEQUENCE LENGTH: 25
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..25
3o IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID NO: 23
GTTATAGACT CCCTGTTCTC ACTGC 25
SEQ ID N0: 24:
SEQUENCE LENGTH: 25
SEQUENCE TYPE: nucleic acid
CA 02282557 1999-08-27
91
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..25
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 24
AGACTCCCTG TTCTCACTGC TTATG 25
0
SEQ ID N0: 25:
SEQUENCE LENGTH: 27
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..27
2o IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 25
ATCTTCACTG CTCACATCGG GGGTAAT 27
SEQ ID N0: 26:
SEQUENCE LENGTH: 23
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
3o FEATURE:
NAME/KEY: primer bind
LOCATION: 1..23
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID NO: 26
TCACTGCTCA CATCGGGGGT AAT 23
CA 02282557 1999-08-27
92
SEQ ID N0: 27:
SEQUENCE LENGTH: 1703
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: double
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (cDNA containing 5~-end nucleotide
sequence)
ORIGINAL SOURCE:
ORGANISM: Mus
ioFEATURE:
NAME/gEY: CDS
LOCATION: 10..1703
IDENTIFICATION
METHOD:
E
SEQUENCE N0:27
DESCRIPTION:
SEQ
ID
i5GATGATCTC
ATG GCA ATG GCTGAACAC CCGGAAGGACAT CATGATAGT 51
AAG
Met Ala Met AlaGluHis ProGluGlyHis HisAspSer
Lys
1 5 10
GCT CTA CAC ATGCTCTAT ACAGCCATTGCC TTTTTAGGG 93
ACT
2oAla Leu His MetLeuTyr ThrAlaThrAla PheLeuGly '
Thr
20 25
GTG GCA CAC AAGGGTGGA GTACTCTTGCTG GTGCTGGCT 135
GAT
Val Ala His LysGlyGly ValLeuLeuLeu ValLeuAla
Asp
30 35 40
25TTA TGT AAA GTTGGTTTT CATACGGCTTCC AGAAAGCTC 177
TGT
Leu Cys Lys ValGlyPhe HisThrAlaSer ArgLysLeu
Cys
45 50 55
TCT ATA GTT GGTGGAGCT AAGCGCCTTCAG GCCTTATCT 219
GAT
Ser Ile Val GlyGlyAla LysArgLeuGln AlaLeuSer
Asp
30 60 65 70
CAG CTT TCT GTGTTTCTC CTGTGCCCATGG GTGATTGTC 261
GTT
Gln Leu Ser ValPheLeu LeuCysProTrp ValIleVal
Val
75 80
CTT TCT ACA ACTGAAAGT AAGGTTGAGTCT TGGTTCTCT 303
GTG
35Leu Ser Thr ThrGluSer LysValGluSer TrpPheSer
Val
85 90 95
CA 02282557 1999-08-27
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CTC ATC ATGCCTTTC ACCACAGTC ATCTTTTTT GTCATGATC 345
Leu Ile MetProPhe ThrThrVal IlePhePhe ValMetIle
100 105 110
CTG GAT TTCTATATG GATTCTGTT TGTTCAGTC AAAATGGAC 387
Leu Asp PheTyrMet AspSerVal CysSerVal LysMetAsp
115 120 125
GTG TCC AAATGTGCC CGCTATGGG TCCTTTCCC ATTTTTATT 429
Val Ser LysCysAla ArgTyrGly SerPhePro IlePheIle
130 135 140
to AGT GCC CTCCTGTTT CGAAATTTC TGGACACAC CCAATAACA 471
Ser Ala LeuLeuPhe GlyAsnPhe TrpThrHis ProIleThr
145 150
GAC CAA CTCCGGGCT ATGAACAGA GCAGCACAC CAGGAGAGC 513
Asp Gln LeuArgAla MetAsnArg AlaAlaHis GlnGluSer
155 160 165
ACA GAA CACGTCCTG TCTGGAGGA GTGGTAGTG AGCGCTGTG 555
Thr Glu HisValLeu SerGlyGly ValValVal SerAlaVal
170 175 180
TTC TTC ATTTTGTCG GCCAACATT CTATCATCT CCCTCTAAG 597
2o Phe Phe IleLeuSer AlaAsnIle LeuSerSer ProSerLys
185 190 195
AGA GGA CAGAAAGGT ACCCTTATT GGATATTCT CCTGAAGGA 639
Arg Gly GlnLysGly ThrLeuIle GlyTyrSer ProGluGly
200 205 210
ACA CCA CTCTATCAC TTCATGGGG GACGCTTTT CAGCACAGC 681
Thr Pro LeuTyrHis PheMetGly AspAlaPhe GlnHisSer
215 220
TCT CAG TCGGTGCCT AGGTTCATT AAGGACTCA CTAAAGCAG 723
Ser Gln SerValPro ArgPheIle LysAspSer LeuLysGln
225 230 235
GTT CTC GAGGAGAGC GACTCTAGG CAGATCTTT TACTTCTTG 765
Val Leu GluGluSer AspSerArg GlnIlePhe TyrPheLeu
240 245 250
TGC TTG AATCTGCTT TTTACCTTT GTGGAGTTG TTCTATGGC 807
Cys Leu AsnLeuLeu PheThrPhe ValGluLeu PheTyrGly
255 260 265
CA 02282557 1999-08-27
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GTG CTA ACCAACAGTCTA GGCCTGATC TCAGATGGA TTTCAC849
Val Leu ThrAsnSerLeu GlyLeuIle SerAspGly PheHis
270 275 280
ATG CTC TTTGACTGCTCG GCTTTGGTC ATGGGACTG TTTGCT891
Met Leu PheAspCysSer AlaLeuVal MeyGlyLeu PheAla
285 290
GCC CTG ATGAGCCGCTGG AAAGCCACC CGGATTTTC TCCTAT933
Ala Leu MetSerArgTrp LysAlaThr ArgIlePhe SerTyr
295 300 305
to GGG TAT GGCCGAATAGAG ATTCTCTCT GGCTTTATT AATGGG975
Gly Tyr GlyArgIleGlu IleLeuSer GlyPheIle AsnGly
310 315 320
CTT TTT CTGATCGTGATA GCATTTTTT GTGTTTATG GAATCA1017
Leu Phe LeuIleValIle AlaPhePhe ValPheMet GluSer
325 330 335
GTG GCT AGACTGATCGAT CCTCCGGAA CTAGACACA AACATG1059
Val Ala ArgLeuIleAsp ProProGlu LeuAspThr AsnMet
340 345 350
CTG ACA CCAGTTTCCGTC GCAGGGCTG ATAGTAAAC CTTATT1101
2o Leu Thr ProValSerVal GlyGlyLeu IleValAsn LeuIle
355 360
GGT ATC TGTGCCTTCAGC CACGCCCAC AGCCATGGC CATGGC1143
Gly Ile CysAlaPheSer HisAlaHis SerHisGly HisGly
365 370 375
GCT TCT CAAGGAAACTGC CACTCTGAT CACGGCCAT TCACAC1185
Ala Ser GlnGlyAsnCys HisSerAsp HisGlyHis SerHis
380 385 390
CAT GCA CATGGACACGGC CATGATCAC GGTCACAGC CACGGC1227
His Ala HisGlyHisGly HisAspHis GlyHisSer HisGly
395 400 405
TTC ACG GGTGGAGGCATG AATGCGAAC ATGAGGGGT GTATTT1269
Phe Thr GlyGlyGlyMet AsnAlaAsn MetArgGly ValPhe
410 415 420
CTC CAT GTGTTGGCAGAC ACACTGGGC AGCATCGGC GTGATT1311
Leu His ValLeuAlaAsp ThrLeuGly SerIleGly ValIle
425 430
CA 02282557 1999-08-27
GTG TCC ACAGTT CTCATAGAG CAGTTTGGATGG TTCATTGCT 1353
Val Ser ThrVal LeuIleGlu GlnPheGlyTrp PheIleAla
435 440 445
GAT CCC CTGTGT TCTCTTTTT ATTGCCGTGTTG ATATTTCTC 1395
5 Asp Pro LeuCys SerLeuPhe IleAlaValLeu IlePheLeu
450 455 460
AGT GTG ATCCCA CTGATTAAA GATGCCTGTCAA GTTCTACTT 1437
Ser Val IlePro LeuIleLys AspAlaCysGln ValLeuLeu
465 470 475
io CTG AGA CTACCA CCTGACCAT GAAAAAGAACTG CATATTGCT 1479
Leu Arg LeuPro ProAspHis GluLysGluLeu HisIleAla
480 485 490
TTA GAA AAGATA CAGAAAATT GAGGGATTAATA TCATACCGA 1521
Leu Glu LysIle GlnLysIle PheGlyLeuIle SerTyrArg
15 495 500
GAC CCT CATTTT TGGCGCCAT TCTGCCAGTATT GTAGCGGGA 1563
Asp Pro HisPhe TrpArgHis SerAlaSerIle ValAlaGly
505 510 515
ACA ATT CATATA CAAGTGACA TCTGAGGTGCTG GAGCAGAGA 1605
2o Thr Ile HisIle GlnValThr SerGluValLeu GluGlnArg
520 525 530
ATT GTA CAGCAG GTTACAGGG ATACTTAAAGAT GCAGGAGTA 1647
Ile Val GlnGln ValThrGly IleLeuLysAsp AlaGlyVal
535 540 545
25 AAC AAC CTAACC ATACAAGTG GAAAAGGAGGCA TACTTTCAG 1689
~
Asn Asn LeuThr IleGlnVal GluLysGluAla TyrPheGln
550 555 560
CAT ATG TCCGGC CT 1703
His Met SerGly
SEQ ID N0: 28:
SEQUENCE LENGTH: 564
SEQUENCE TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: protein
SEQUENCE DESCRIPTION: SEQ ID N0: 28
CA 02282557 1999-08-27
96
Met Ala Lys Met Ala Glu His Pro Glu Gly His His Asp Ser
1 5 10
Ala Leu Thr His Met Leu Tyr Thr Ala Thr Ala Phe Leu Gly
15 20 25
Val Ala Asp His Lys Gly Gly Val Leu Leu Leu Val Leu Ala
30 35 40
Leu Cys Cys Lys Val Gly Phe His Thr Ala Ser Arg Lys Leu
45 50 55
Ser Ile AspValGly GlyAlaLysArg LeuGlnAla LeuSer
l0 60 65 70
Gln Leu ValSerVal PheLeuLeuCys ProTrpVal IleVal
75 80
Leu Ser ValThrThr GluSerLysVal GluSerTrp PheSer
85 90 95
~5 Leu Ile MetProPhe ThrThrValIle PhePheVal MetIle
100 105 110
Leu Asp PheTyrMet AspSerValCys SerValLys MetAsp
115 120 125
Val Ser LysCysAla ArgTyrGlySer PheProIle PheIle
20 130 135 140
Ser Ala LeuLeuPhe GlyAsnPheTrp ThrHisPro IleThr
145 150
Asp Gln LeuArgAla MetAsnArgAla AlaHisGln GluSer
155 160 165
25 Thr Glu HisValLeu SerGlyGlyVal ValValSer AlaVal
170 175 180
Phe Phe IleLeuSer AlaAsnIleLeu SerSerPro SerLys
185 190 195
Arg Gly GlnLysGly ThrLeuIleGly TyrSerPro GluGly
30 200 205 210
Thr Pro LeuTyrHis PheMetGlyAsp AlaPheGln HisSer
215 220
Ser Gln SerValPro ArgPheIleLys AspSerLeu LysGln
225 230 235
35 Val Leu GluGluSer AspSerArgGln IlePheTyr PheLeu
240 245 250
CA 02282557 1999-08-27
97
Cys Leu AsnLeuLeu PheThrPhe ValGluLeu PheTyrGly
255 260 265
Val Leu ThrAsnSer LeuGlyLeu IleSerAsp GlyPheHis
270 275 280
Met Leu PheAspCys SerAlaLeu ValMeyGly LeuPheAla
285 290
Ala Leu MetSerArg TrpLysAla ThrArgIle PheSerTyr
295 300 305
Gly Tyr GlyArgIle GluIleLeu SerGlyPhe IleAsnGly
io 310 315 320
Leu Phe LeuIleVal IleAlaPhe PheValPhe MetGluSer
325 330 335
Val Ala ArgLeuIle AspProPro GluLeuAsp ThrAsnMet
340 345 350
~5 Leu Thr ProValSer ValGlyGly LeuIleVal AsnLeuIle
355 360
Gly Ile CysAlaPhe SerHisAla HisSerHis GlyHisGly
365 370 375
Ala Ser GlnGlyAsn CysHisSer AspHisGly HisSerHis
20 380 385 390
His Ala HisGlyHis GlyHisAsp HisGlyHis SerHisGly
395 400 405
Phe Thr GlyGlyGly MetAsnAla AsnMetArg GlyValPhe
410 415 420
25 Leu His ValLeuAla AspThrLeu GlySerIle GlyValIle
425 430
Val Ser ThrValLeu IleGluGln PheGlyTrp PheIleAla
435 440 445
Asp Pro LeuCysSer LeuPheIle AlaValLeu IlePheLeu
30 450 455 460
Ser Val IleProLeu IleLysAsp AlaCysGln ValLeuLeu
465 470 475
Leu Arg LeuProPro AspHisGlu LysGluLeu HisIleAla
480 485 490
35 Leu Glu LysIleGln LysIlePhe GlyLeuIle SerTyrArg
495 500
CA 02282557 1999-08-27
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Asp Pro His Phe Trp Arg His Ser Ala Ser Ile Val Ala Gly
505 510 515
Thr Ile His Ile Gln Val Thr Ser Glu Val Leu Glu Gln Arg
520 525 530
Ile Val Gln Gln Val Thr Gly Ile Leu Lys Asp Ala Gly Val
535 540 545
Asn Asn Leu Thr Ile Gln Val Glu Lys Glu Ala Tyr Phe Gln
550 555 560
His Met Ser Gly
0
SEQ ID N0: 29:
SEQUENCE LENGTH: 29
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..29
2o IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 29
GGGAATTCAT GGGTAAGACC CTGCGGGGC 29
SEQ ID N0: 30:
SEQUENCE LENGTH: 35
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
3o FEATURE: .
NAME/KEY: primer bind
LOCATION: 1..35
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 30
GGAAGCTTTC ACTCATGTAA CCCAGATTCT CCAGC 35
CA 02282557 1999-08-27
99
SEQ ID N0: 31:
SEQUENCE LENGTH: 35
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE
NAME/KEY: primer bind
LOCATION: 1 .. 35
io IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 31
GGTTTTCTAG AGCTCGTGCA GACCTGGAGA CGGGC 35
SEQ ID N0: 32:
i5 SEQUENCE LENGTH: 39
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
2o FEATURE:
NAME/KEY: primer bind
LOCATION: 1..39
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID NO: 32
25 GGGTTTTCTA GATCTTCAGA AATGAGGGCG ACTTTTGTG 39
SEQ ID NO: 33:
SEQUENCE LENGTH: 29
SEQUENCE TYPE: nucleic acid
3o STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
35 LOCATION: 1..29
IDENTIFICATION METHOD: E
CA 02282557 1999-08-27
Z00
SEQUENCE DESCRIPTION: SEQ ID N0: 33
GGGATCCGCA TAGTACAGCA GGTTACAGG 29
SEQ ID N0: 34:
SEQUENCE LENGTH: 29
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
to FEATURE:
NAME/KEY: primer bind
LOCATION: 1..29
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 34
~5 GGTCGACCCT TTAAAATACA TAACTCAAA 29
SEQ ID N0: 35:
SEQUENCE LENGTH: 30
SEQUENCE TYPE: nucleic acid
2o STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
25 LOCATION: 1..30
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 35
AATGTA~GCA GTATGGAAAT GGAAAGTTGC 30
3o SEQ ID N0: 36:
SEQUENCE LENGTH: 28
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
35 MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
CA 02282557 1999-08-27
ZO1
NAME/KEY: primer bind
LOCATION: 1..28
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 36
CGAATGGGCT GACCGCTTCC TCGTGCTT 28
SEQ ID N0: 37:
SEQUENCE LENGTH: 30
SEQUENCE TYPE: nucleic acid
to STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
i5 LOCATION: 1..30
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 37
CGGCAGGAGC AAGGTGAGAT GACAGGAGAT 30
2o SEQ ID N0: 38:
SEQUENCE LENGTH: 30
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
25 MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..30
IDENTIFICATION METHOD: E
3o SEQUENCE DESCRIPTION: SEQ ID N0: 38
TTACAGTGTC AGGAATAAAG GCTATGCTTC 30
SEQ ID N0: 39:
SEQUENCE LENGTH: 20
35 SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
CA 02282557 1999-08-27
102
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..20
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 39
GACTCGATCT CATGGCAAAG 20
o SEQ ID N0: 40:
SEQUENCE LENGTH: 21
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..21
IDENTIFICATION METHOD: E
2o SEQUENCE DESCRIPTION: SEQ ID N0: 40
TAGAGCATGT GAGTTAGAGC A 21
SEQ ID N0: 41:
SEQUENCE LENGTH: 21
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
3o NAME/KEY: primer bind
LOCATION: 1..21
IDENTIFICATION METHOD: E
SEQUENCE DESCRIPTION: SEQ ID N0: 41
CCAACATTCT ATCATCTCCC T 21
SEQ ID N0: 42:
CA 02282557 1999-08-27
103
SEQUENCE LENGTH: 19
SEQUENCE TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: other nucleic acid (synthetic DNA)
FEATURE:
NAME/KEY: primer bind
LOCATION: 1..19
IDENTIFICATION METHOD: E
to SEQUENCE DESCRIPTION: SEQ ID N0: 42
AGCGGCTCAT CAGGGCAGC ig
